Difference between revisions of "Team:ATOMS-Turkiye/Extras/Notebook"

Line 255: Line 255:
  
  
−
<h2 class="scroll">July</h2>
+
 
 +
<h2 class="scroll">August</h2>
 
<div id="content-div">
 
<div id="content-div">
  
−
<p>01.07. Some preparetions were made for experiments.</p>
+
<p>11.08. We started designing our wiki.</p>
−
<p>05.07. Today is the first day of shooting. We just learned picking costumes isn’t as easy as we think.</p>
+
<p>18.08.2015 We got a little tired of doing experiments, so treated ourselves with an awesome team dinner at a fish restaurant.</p>
−
<p>06.07. If there is a film, then there is After Effects work to do. Good luck with this, dear teammate Şahika.</p>
+
<p>22.08.2015 A team dinner again, but this time our dear professors Mehmet and Esra Gündüz hosted us at their house.</p>
−
<p>08.07. We made a presentation to the pre-med students which came from USA for intership and they loved our project.</p>
+
<p>29.08.2015 Wiki’s tour part is drawn, now it’s time to color these drawings!</p>
−
<p>21.07. Drawing pictures for wiki began. Thus we discovered our teammate Kevser’s hidden talents.</p>
+
−
<p>22.07. Our gene blocks has just arrived. Let the experiments begin! We are all ready now.</p>
+
−
<p>29.07. We released our first video of Virtual Hospital. Also arm’s design is finished.</p>
+
 
</div>
 
</div>
  
−
    <a href="#" class="clickme" id="toggle" onclick="toggle_visibility('2');">Week 2<img src="https://static.igem.org/mediawiki/2014/f/f8/LMU14_arrow_down.png" id="2" class="tiger"></a>
+
  <a href="#" class="clickme" id="toggle" onclick="toggle_visibility('6');">Week 6<img src="https://static.igem.org/mediawiki/2014/f/f8/LMU14_arrow_down.png" id="6" class="tiger"></a>
  
  
 
<div class="box">
 
<div class="box">
  
−
 
+
Mini Prep of “pSB1C3-RFP”
−
Gradient PCR of „pCAG with CAG FWD/CAG  REV. Primers/pCMV REV.” (01.07.2015)
+
 
</html>
 
</html>
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!Gradient PCR from pCAGGS (CAG FWD – CAG REVERSE)
+
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
 
|-
 
|-
−
|||MgCl₂||(NH₄)2SO₄||VR fwd||VR rv||dNTP||Tag||ddH₂O||DNA||Total
+
|1||blank||biospec||8/1/2015 6:03:37 PM||-0.1||ng/ul||-0.003||-0.009||0.30||0.12||DNA||50.00
 
|-
 
|-
−
|1x||2.5 ul||2.5 ul||0.5 ul||0.5 ul||0.5 ul||0.2 ul||16.3 ul||2.0 ul||25.0 ul
+
|2||psb1c3-A||biospec||8/1/2015 6:06:00 PM||207.0||ng/ul||4.140||2.184||1.90||2.11||DNA||50.00
 
|-
 
|-
−
|9X||22.5 ul||22.5 ul||4.5 ul||4.5 ul||4.5 ul||1.8 ul||146.7 ul||18.0 ul||225.0 ul
+
|3||psb1c3-B||biospec||8/1/2015 6:07:07 PM||209.0||ng/ul||4.179||2.210||1.89||2.19||DNA||50.00
 +
|-
 +
|4||psb1c3-C||biospec||8/1/2015 6:08:05 PM||157.3||ng/ul||3.147||1.664||1.89||2.09||DNA||50.00
 +
|-
 +
|5||psb1c3-D||biospec||8/1/2015 6:08:58 PM||242.5||ng/ul||4.851||2.548||1.90||2.11||DNA||50.00
 +
|-
 +
|6||psb1c3-E||biospec||8/1/2015 6:09:42 PM||116.4||ng/ul||2.329||1.215||1.92||2.17||DNA||50.00
 +
|-
 +
|7||psb1c3-F||biospec||8/1/2015 6:10:28 PM||236.4||ng/ul||4.729||2.503||1.89||2.17||DNA||50.00
 +
|-
 +
|8||psb1c3-71||biospec||8/1/2015 6:11:33 PM||111.7||ng/ul||2.233||1.175||1.90||2.10||DNA||50.00
 +
|-
 +
|9||psb1c3-72||biospec||8/1/2015 6:12:14 PM||106.4||ng/ul||2.127||1.100||1.93||2.23||DNA||50.00
 +
|-
 +
|10||psb1c3-8||biospec||8/1/2015 6:12:51 PM||90.7||ng/ul||1.814||0.959||1.89||1.98||DNA||50.00
 
|}
 
|}
−
<html>
 
−
 
−
<p>60-68˚C</p>
 
−
<a href="https://static.igem.org/mediawiki/2015/f/f9/ATOMS-Turkiye_cag_reverse.jpeg"  data-lightbox="image-1" class="img="left"><img src="https://static.igem.org/mediawiki/2015/f/f9/ATOMS-Turkiye_cag_reverse.jpeg" width="390" height="250"></a>
 
−
<p>Results weren’t matching with expected results, experiment will be repeated.</p>
 
  
 +
<html>
 
<br>
 
<br>
  
 +
Ligation of „pSB1C3 – Gblocks“
 
</html>
 
</html>
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!Gradient PCR from pCAGGS (CAG FWD – Chicken β Aktin REVERSE)
+
!!!2!!3!!5!!6!!7!!8!!9!!10!!11!!12!!13!!14!!15!!
 
|-
 
|-
−
|||MgCl₂||(NH₄)2SO₄||VR fwd||VR rv||dNTP||Tag||ddH₂O||DNA||Total
+
|Insert||7.3 ul||7.0 ul||4.8 ul||8.4 ul||5.8 ul||8.3 ul||3.6 ul||3.9 ul||3.4 ul||4.4 ul||5.9 ul||3.9 ul||5.9 ul||
 
|-
 
|-
−
|1x||2.5 ul||2.5 ul||0.5 ul||0.5 ul||0.5 ul||0.2 ul||16.3 ul||2.0 ul||25.0 ul
+
|Vector||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||
 
|-
 
|-
−
|9X||22.5 ul||22.5 ul||4.5 ul||4.5 ul||4.5 ul||1.8 ul||146.7 ul||18.0 ul||225.0 ul
+
|Buffer||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||
 +
|-
 +
|Enzyme||1.0 ul||1.0 ul||1.0 ul||1.0 ul||1.0 ul||1.0 ul||1.0 ul||1.0 ul||1.0 ul||1.0 ul||1.0 ul||1.0 ul||1.0 ul||
 +
|-
 +
|ddH₂O||7.7 ul||8.0 ul||10.2 ul||6.6 ul||9.2 ul||6.7 ul||11.4 ul||11.1 ul||11.6 ul||10.6 ul||9.1 ul||11.1 ul||9.1 ul||
 +
|-
 +
|Total||20.0 ul||20.0 ul||20.0 ul||20.0 ul||20.0 ul||20.0 ul||20.0 ul||20.0 ul||20.0 ul||20.0 ul||20.0 ul||20.0 ul||20.0 ul||
 
|}
 
|}
 +
 +
 
<html>
 
<html>
−
<p>60-68˚C</p>
+
</div>
−
<a href="https://2015.igem.org/File:ATOMS-Turkiye_chicken.jpeg"  data-lightbox="image-1" class="img="left"><img src="https://2015.igem.org/File:ATOMS-Turkiye_chicken.jpeg" width="390" height="250"></a>
+
−
<p>Results weren’t matching with expected results, experiment will be repeated.</p>
+
  
−
<br>
+
      <a href="#" class="clickme" id="toggle" onclick="toggle_visibility('7');">Week 7<img src="https://static.igem.org/mediawiki/2014/f/f8/LMU14_arrow_down.png" id="6" class="tiger"></a>
 +
<div class="box">
  
−
Protocols of „Phusion Pol, and Q5 Polymerase” (02.07.2015)
+
Colony PCR of „pSB1C3 – Gblocks“ (03.08.2015)
 
</html>
 
</html>
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!Phusion DNA Polymerase
+
!Colony PCR
 
|-
 
|-
−
|||dNTP||Fwd primer||Rev primer||Buffer||Phusion Pol.||ddH₂O||DNA||Total
+
|||MgCl₂||(NH₄)2SO₄||VR fwd||VR rv||dNTP||Tag||ddH₂O||DNA||Total
 
|-
 
|-
−
|1x||0.4 ul||2.0 ul||2.0 ul||4.0 ul||0.2 ul||10.4 ul||1.0 ul||20.0 ul
+
|1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul
 +
|-
 +
|22X||55.0 ul||55.0 ul||22.0 ul||22.0 ul||11.0 ul||4.4 ul||271.0 ul||||440.4 ul
 
|}
 
|}
  
Line 322: Line 339:
 
!Cycling
 
!Cycling
 
|-
 
|-
−
|||98˚C||98˚C||56˚C||72˚C||72˚C||Cycle
+
|||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle
 
|-
 
|-
−
|Time||2’||10’’||30’’||1’||5’||35x
+
|Time||5’||30’’||30’’||1.5’||5’||35x
 
|}
 
|}
−
<html>
+
<a href="https://static.igem.org/mediawiki/2015/9/92/ATOMS-Turkiye_03_08_colony_pcr.jpeg" data-lightbox="image-1" class="img="left"><img src="https://static.igem.org/mediawiki/2015/9/92/ATOMS-Turkiye_03_08_colony_pcr.jpeg" width="410" height="340"></a>
 +
<p>Result: negative</p>
  
 
<br>
 
<br>
  
−
</html>
+
Colony PCR of „pSB1C3 – Gblocks #14“ (03.08.2015)
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!Q5 DNA Polymerase
+
!Colony PCR
 
|-
 
|-
−
|||dNTP||Fwd primer||Rev primer||Buffer||Q5 Pol.||ddH₂O||DNA||Total
+
|||MgCl₂||(NH₄)2SO₄||VR fwd||VR rv||dNTP||Tag||ddH₂O||DNA||Total
 +
|-
 +
|1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul
 
|-
 
|-
−
|1x||0.5 ul||2.5 ul||2.5 ul||5.0 ul||0.25 ul||8.25 ul||1.0 ul||20.0 ul
+
|36X||90.0 ul||90.0 ul||36.0 ul||36.0 ul||18.0 ul||7.2 ul||442.8 ul||||720.0 ul
 
|}
 
|}
  
Line 342: Line 362:
 
!Cycling
 
!Cycling
 
|-
 
|-
−
|||98˚C||98˚C||56˚C||72˚C||72˚C||Cycle
+
|||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle
 
|-
 
|-
−
|Time||2’||10’’||30’’||1’||5’||35x
+
|Time||5’||30’’||30’’||1.5’||5’||35x
 
|}
 
|}
−
<html>
+
<a href="https://static.igem.org/mediawiki/2015/1/19/ATOMS-Turkiye_03_08_colony_pcr_14.jpeg" data-lightbox="image-1" class="img="left"><img src="https://static.igem.org/mediawiki/2015/1/19/ATOMS-Turkiye_03_08_colony_pcr_14.jpeg" width="410" height="360"></a>
−
<p>Defterde jel görüntüsü yok.</p>
+
<a href="https://static.igem.org/mediawiki/2015/b/bf/ATOMS-Turkiye_03_08_colony_pcr_14_1.jpeg" data-lightbox="image-1" class="img="left"><img src="https://static.igem.org/mediawiki/2015/b/bf/ATOMS-Turkiye_03_08_colony_pcr_14_1.jpeg" width="360" height="360"></a>
−
</div>
+
<p>Result: negative</p>
  
−
    <a href="#" class="clickme" id="toggle" onclick="toggle_visibility('3');">Week 3<img src="https://static.igem.org/mediawiki/2014/f/f8/LMU14_arrow_down.png" id="3" class="tiger"></a>
+
<br>
  
−
<div class="box">
+
Digestion of “pSB1C3-RFP” (03.08.2015)
−
 
+
−
Gradient PCR of „pCAG with CAG-FWD/CAG REV. Primers and Phusion Pol.”
+
−
</html>
+
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!Gradient PCR from pCAGGS
+
!Digestion of “pSB1C3-RFP”
 
|-
 
|-
−
|||dNTP||Fwd primer||Rev primer||Buffer||Phusion Pol.||ddH₂O||DNA||Total
+
|||Vector||Fast Digest Buffer||EcoRI (FD)||PstI (FD)||ddH₂O||Total
 
|-
 
|-
−
|1x||0.4 ul||2.0 ul||2.0 ul||4.0 ul||0.2 ul||10.4 ul||1.0 ul||20.0 ul
+
|1x||2.0 ul||2.0 ul||1.0 ul||1.0 ul||14.0 ul||20.0 ul
 
|}
 
|}
−
<html>
+
<p>37˚C 2h</p>
−
<p>62-64˚C</p>
+
<a href="https://static.igem.org/mediawiki/2015/d/df/ATOMS-Turkiye_03_08_rfp_kesim.jpeg" data-lightbox="image-1" class="img="left"><img src="https://static.igem.org/mediawiki/2015/d/df/ATOMS-Turkiye_03_08_rfp_kesim.jpeg" width="410" height="360"></a>
−
<a href="https://static.igem.org/mediawiki/2015/c/ce/ATOMS-Turkiye_07%2C07.jpeg" data-lightbox="image-1" class="img="left"><img src="https://static.igem.org/mediawiki/2015/c/ce/ATOMS-Turkiye_07%2C07.jpeg" width="390" height="250"></a>
+
<p>Gel Electropheresis was made.</p>
−
<p>Result: Gel extraction was performed. PCR (+): 680 bp</p>
+
<p>Result: Bands were at the expected section. Gel purification was made.</p>
 +
 
  
 
<br>
 
<br>
  
−
 
+
Ligation of “pSB1C3 – Gblock #14”
−
</html>
+
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
+
!Ligation of “pSB1C3 – Gblock #14”
 
|-
 
|-
−
|1||eb||biospec||7/8/2015 1:56:43 AM||0.1||ng/ul||0.002||-0.009||-0.25||-0.19||DNA||50.00
+
|||Insert||Vector||T4 DNA Ligase Buffer||T4 DNA Ligase||ddH₂O||Total
 
|-
 
|-
−
|2||pCAG||biospec||7/8/2015 1:58:14 AM||13.7||ng/ul||0.275||0.138||1.98||0.15||DNA||50.00
+
|1) PCR clean up vektörü ile 2.5 ul||2.5 ul||2.0 ul
 +
|-
 +
|(100ng)||2.0 ul
 +
|-
 +
|||1.0 ul||12.5 ul||20.0 ul
 +
|-
 +
|2) Gel Extraction 7.5 ul||7.5 ul||0.8 ul
 +
|-
 +
|(50ng)||2.0 ul
 +
|-
 +
|||1.0 ul||8.7 ul||20.0 ul
 
|}
 
|}
−
<html>
 
  
 
<br>
 
<br>
  
−
Creating “Plasmid pCAG” via “pTRE” and “Promoter pCAG”
 
−
</html>
 
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!Digestion of “pTRE” and “Promoter pCAG”
+
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
 
|-
 
|-
−
|||pTRE (1536 ng/ul)||pCAG (promoter) (13.7 ng/ul)||EcoRI||XhoI ||Neb 3.1 Buffer||ddH₂O||Total
+
|1||blank||biospec||8/3/2015 6:10:02 PM||0.2||ng/ul||0.004||-0.010||-0.41||1.13||DNA||50.00
 
|-
 
|-
−
|1||3.2 ul||-||0.5 ul||0.5 ul||2.0 ul||13.8 ul||20.0 ul
+
|2||psb1c3 gel||biospec||8/3/2015 6:12:16 PM||62.5||ng/ul||1.250||0.664||1.88||0.90||DNA||50.00
 
|-
 
|-
−
|2||-||10.0 ul||0.5 ul||0.5 ul||2.0 ul||7.0 ul||20.0 ul
+
|4||psb1c3 clean-up||biospec||8/3/2015 6:13:39 PM||170.5||ng/ul||3.409||1.884||1.81||1.33||DNA||50.00
 
|}
 
|}
−
<html>
 
−
<p>pTRE-delta-TRE was made after digestion.</p>
 
−
<p>Concentration: 99.9 ng/ul</p>
 
−
<p>The final concentration of pCAG is 6.855 ng/ul.</p>
 
  
 
<br>
 
<br>
  
−
 
+
PCR of “G-Bloks via Q5 Polymerase” (04.08.2015)
−
</html>
+
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!Ligation of „pTRE  TRE“ and „Digested promoter pCAG“
+
!PCR!!!!!!!!!!!!
 
|-
 
|-
−
|
+
|||SV40 poly A rev||CMV fwd||tetR rev||2x Q5 Master Mix||DNA||Total
 
|-
 
|-
−
|||pTRE  TRE
+
|1x||2.5 ul||2.5 ul||2.5 ul||10.0 ul||5.0 ul||20.0 ul
 +
|}
 +
 
 +
<br>
 +
 
 +
{| border="1" class="wikitable"
 +
!Cycling SV40 rev/CMV fwd
 
|-
 
|-
−
|pCAG ||T4 DNA Ligase||Buffer||ddH₂O||Total||
+
|||95˚C||95˚C||63˚C||72˚C||72˚C||Cycle
 
|-
 
|-
−
|1:1||3.0 ul||7.0 ul||0.5 ul||2.0 ul||7.5 ul||20.0 ul||
+
|Time||5’||30’’||30’’||1’||2’||35x
 
|}
 
|}
−
<html>
 
−
 
−
<p>Ligation products were transformed into E.Coli/BL321 strain.</p>
 
−
<p>Result: No colonies were observed.</p>
 
  
 
<br>
 
<br>
  
−
Digestion of “pTEToff and pET45 Vectors”
 
−
</html>
 
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!Digestion of “pTEToff  and pET45 Vectors”
+
!Cycling CMV fwd/tetR rev
 
|-
 
|-
−
|||pTEToff (1141 ng/ul)||pET45 (485 ng/ul)||XhoI (Neb)||BamHI (Neb)||SalI (Thermo)||HindIII (Thermo)||Cut Smart Buffer||Fast Digest Buffer||ddH₂O||Total||
+
|||98˚C||98˚C||66˚C||72˚C||72˚C||Cycle
 
|-
 
|-
−
|1||3.1 ul||-||-||-||0.5 ul||0.5 ul||-||2.0 ul||13.9 ul||20.0 ul||37˚C 1h
+
|Time||30’’||10’’||30’’||1’||2’||35x
 +
|}
 +
 
 +
<br>
 +
 
 +
Digestion of “pSB1C3-RFP” (04.08.2015)
 +
{| border="1" class="wikitable"
 +
!Digestion!!!!!!!!!!!!
 +
|-
 +
|||Vector||Cut Smart Buffer||EcoRI||SpeI||ddH₂O||Total
 
|-
 
|-
−
|2||-||4.1 ul||0.5 ul||0.5 ul||-||-||2.0 ul||-||12.9 ul||20.0 ul||37˚C 2h
+
|1x||2.0 ul||2.0 ul||1.0 ul||1.0 ul||14.0 ul||20.0 ul
 
|}
 
|}
−
<html>
+
 
−
<a href="https://static.igem.org/mediawiki/2015/e/e6/ATOMS-Turkiye_10%2C07.jpeg" data-lightbox="image-1" class="img="left"><img src="https://static.igem.org/mediawiki/2015/e/e6/ATOMS-Turkiye_10%2C07.jpeg" width="250" height="350"></a>
+
<p>37˚C 4h</p>
−
<p>Result: Bands were at the expected section. Gel extraction was made.</p>
+
<a href="https://static.igem.org/mediawiki/2015/8/84/ATOMS-Turkiye_04_08_15.jpeg" data-lightbox="image-1" class="img="left"><img src="https://static.igem.org/mediawiki/2015/8/84/ATOMS-Turkiye_04_08_15.jpeg" width="410" height="360"></a>
 +
<p>Result: Bands were at the expected section.</p>
  
 
<br>
 
<br>
  
−
</html>
 
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
 
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
 
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
 
|-
 
|-
−
|1||eb||biospec||7/10/2015 11:37:54 PM||-0.4||ng/ul||-0.008||-0.015||0.58||-0.39||DNA||50.00
+
|1||blank||biospec||8/4/2015 7:03:09 AM||0.9||ng/ul||0.017||0.005||3.41||0.68||DNA||50.00
 
|-
 
|-
−
|2||pET45 x+b||biospec||7/10/2015 11:40:59 PM||59.0||ng/ul||1.180||0.612||1.93||0.74||DNA||50.00
+
|2||Blank2||biospec||8/4/2015 7:04:51 AM||-0.5||ng/ul||-0.010||-0.017||0.59||0.45||DNA||50.00
 
|-
 
|-
−
|3||pTEToff s+h||biospec||7/10/2015 11:41:58 PM||55.5||ng/ul||1.110||0.581||1.91||0.25||DNA||50.00
+
|4||clean up||biospec||8/4/2015 7:05:55 AM||86.8||ng/ul||1.735||0.913||1.90||0.40||DNA||50.00
 +
|-
 +
|5||gel ext ||biospec||8/4/2015 7:06:51 AM||36.2||ng/ul||0.723||0.380||1.90||0.29||DNA||50.00
 
|}
 
|}
−
<html>
 
  
 
<br>
 
<br>
  
−
Creating “Plasmid pCAG” via “pTRE” and “Promoter pCAG” – Repeat
+
Colony PCR of “11,12,13 pSB1C3 - GBlocks”
−
</html>
+
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!Digestion of “pTRE with EcoRI/XhoI”
+
!Colony PCR
 
|-
 
|-
−
|||pTRE||XhoI||EcoRI||Neb 2.1 Buffer||ddH₂O||Total||
+
|||MgCl₂||(NH₄)2SO₄||VF||VR||Tag||ddH₂O||DNA||Total||
 
|-
 
|-
−
|1||3.2 ul||0.5 ul||0.5 ul||2.0 ul||13,8 ul||20.0 ul||37˚C 2h
+
|1x||2.5 ul||2.5 ul||0.5 ul||0.5 ul||0.2 ul||13.3 ul||5.0 ul||25.0 ul||
 
|-
 
|-
−
|2||3.2 ul||0.5 ul||0.5 ul||2.0 ul||13,8 ul||20.0 ul||37˚C 2h/0.5 ul CIP/37˚C 30’/50˚C 30’
+
|17X||42.5 ul||42.5 ul||8.5 ul||8.5 ul||3.4 ul||226.1 ul||||340.0 ul||
 
|}
 
|}
−
<html>
 
  
−
<p>Result: Gel extraction was made.</p>
+
{| border="1" class="wikitable"
 +
!Cycling CMV fwd/tetR rev
 +
|-
 +
|||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle
 +
|-
 +
|Time||5’||30’’||30’’||1.5’||5’||35x
 +
|}
 +
<a href="https://static.igem.org/mediawiki/2015/3/30/ATOMS-Turkiye_04_08_15_colony_pcr_11%2C12%2C13.jpeg" data-lightbox="image-1" class="img="left"><img src="https://static.igem.org/mediawiki/2015/3/30/ATOMS-Turkiye_04_08_15_colony_pcr_11%2C12%2C13.jpeg" width="410" height="360"></a>
 +
<p>Result: negative</p>
  
 
<br>
 
<br>
  
−
</html>
+
Ligation and Transformation of “5,6,7,8,9 pSB1C3-GBlocks”
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
+
!Ligation of “GBlocks 11-12 pSB1C3”
 
|-
 
|-
−
|1||eb||biospec||7/9/2015 6:32:17 PM||-0.7||ng/ul||-0.015||-0.022||0.66||0.21||DNA||50.00
+
|||Vector||Gen||Buffer||Ligase||ddH₂O||Total
 
|-
 
|-
−
|2||hre cmv mini||biospec||7/9/2015 6:34:10 PM||13.8||ng/ul||0.277||0.141||1.97||0.03||DNA||50.00
+
|5||0.8 ul||2.37 ul||1.0 ul||0.5 ul||5.33 ul||10.0 ul
 
|-
 
|-
−
|3||ptre delta tre cip -||biospec||7/9/2015 6:34:54 PM||88.7||ng/ul||1.774||0.941||1.88||0.24||DNA||50.00
+
|6||0.8 ul||4.2 ul||1.0 ul||0.5 ul||3.5 ul||10.0 ul
 
|-
 
|-
−
|4||ptre delta tre cip +||biospec||7/9/2015 6:35:35 PM||86.0||ng/ul||1.721||0.947||1.82||0.25||DNA||50.00
+
|7||0.8 ul||2.94 ul||1.0 ul||0.5 ul||4.76 ul||10.0 ul
 +
|-
 +
|8||0.8 ul||4.16 ul||1.0 ul||0.5 ul||3.54 ul||10.0 ul
 +
|-
 +
|9||0.8 ul||1.79 ul||1.0 ul||0.5 ul||5.91 ul||10.0 ul
 
|}
 
|}
−
<html>
+
<p>50ng DNA</p>
 +
<p>1 vector:0.5 insert</p>
 +
<p>Transfomation with TOP10.</p>
  
 
<br>
 
<br>
  
−
Creating “Plasmid pCAG” – Continue
+
Ligation of “GBlocks 11-12 pSB1C3”
−
</html>
+
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!Ligation of „pTRE  TRE“ and „Digested promoter pCAG“
+
!Ligation of “GBlocks 11-12 pSB1C3”
 
|-
 
|-
−
|
+
|||pSB1C3||Insert||Buffer||Ligase||ddH₂O||Total
 
|-
 
|-
−
|||pTRE  TRE CIP (+)||pTRE  TRE CIP (-)||pCAG (6.85 ng/ul)||T4 DNA Ligase||Buffer||ddH₂O||Total
+
|11||0.8 ul||1.7 ul||1.0 ul||0.5 ul||6.0 ul||10.0 ul
 
|-
 
|-
−
|1||3.0 ul||-||7.0 ul||0.5 ul||2.0 ul||7.5 ul||20.0 ul
+
|12||0.4 ul||1.1 ul||1.0 ul||0.5 ul||7.0 ul||10.0 ul
−
|-
+
−
|2||-||3.0 ul||7.0 ul||0.5 ul||2.0 ul||7.5 ul||20.0 ul
+
 
|}
 
|}
−
<html>
+
Transformation was made.
−
<p>Room Temperature 1h</p>
+
−
<p>Result: Transformation was made. No colonies were observed at first plate. At the second plate there was five colonies. Colony PCR will be made.</p>
+
−
</div>
+
  
−
    <a href="#" class="clickme" id="toggle" onclick="toggle_visibility('4');">Week 4<img src="https://static.igem.org/mediawiki/2014/f/f8/LMU14_arrow_down.png" id="4" class="tiger"></a>
+
<br>
  
−
<div class="box">
+
Colony PCR of “5,6,7,8,9,T7 system – Gblocks”
−
 
+
−
Creating “Plasmid pCAG” – Continue
+
−
</html>
+
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!Colony PCR from “pCAG” with “pTRE Luc fwd/SV40 polyA re. primers”
+
!Colony PCR  
 
|-
 
|-
−
|||MgCl₂||(NH₄)2SO₄||pTRE Luc fwd||SV40 rev||dNTP||Tag||ddH₂O||DNA||Total
+
|||MgCl₂||(NH₄)2SO₄||VR||VF||dNTP||Tag||ddH₂O||DNA||Total
 
|-
 
|-
−
|1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul
+
|1x||2.5 ul||2.5 ul||0.5 ul||0.5 ul||0.5 ul||0.1 ul||13.3 ul||5.0 ul||24.9 ul
 
|-
 
|-
−
|6X||15.0 ul||15.0 ul||6.0 ul||6.0 ul||3.0 ul||1.2 ul||73.8 ul||||120.0 ul
+
|58X||145.0 ul||145.0 ul||29.0 ul||29.0 ul||29.0 ul||5.8 ul||771.4 ul||||1154.2 ul
 
|}
 
|}
−
 
+
{| border="1" class="sortable"
−
{| border="1" class="wikitable"
+
 
!Cycling
 
!Cycling
 
|-
 
|-
−
|||95˚C||95˚C||55˚C||72˚C||72˚C||Cycle
+
|||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle
 
|-
 
|-
 
|Time||5’||30’’||30’’||1.5’||5’||35x
 
|Time||5’||30’’||30’’||1.5’||5’||35x
 
|}
 
|}
−
<html>
 
−
<a href="https://static.igem.org/mediawiki/2015/6/6d/ATOMS-Turkiye_plasmid_pcag.jpeg"  data-lightbox="image-1" class="img="left"><img src="https://static.igem.org/mediawiki/2015/6/6d/ATOMS-Turkiye_plasmid_pcag.jpeg" width="250" height="330"></a>
 
−
<p>Result: All bands were observed around 700 bp, results were negative. Ligation will be repeated.</p>
 
−
<p>(+) bant: 923 bp</p>
 
−
<p>(-) bant: 705 bp</p>
 
−
 
  
 
<br>
 
<br>
−
Creating “Plasmid pCAG” – Repeat
+
 
−
</html>
+
Ligation of “GBlocks 2,5,6,7,8,9,10,12,13,15,17 pSB1C3-RFP”
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!Ligation of „pTRE  TRE“ and „Digested Promoter pCAG“
+
!Ligation of “GBlocks 11-12 pSB1C3”
 
|-
 
|-
−
|
+
|||pSB1C3||Insert||Buffer||Ligase||ddH₂O||Total
 
|-
 
|-
−
|||pTRE  TRE CIP (+)||pTRE  TRE CIP (-)||pCAG (6.85 ng/ul)||T4 DNA Ligase||Buffer||ddH₂O||Total
+
|2||1.4 ul||3.65 ul||1.0 ul||0.5 ul||3.45 ul||10.0 ul
 
|-
 
|-
−
|1||3.0 ul||-||2.5 ul||0.5 ul||2.0 ul||12.0 ul||20.0 ul
+
|5||1.4 ul||2.4 ul||1.0 ul||0.5 ul||4.7 ul||10.0 ul
 
|-
 
|-
−
|2||-||3.0 ul||2.5 ul||0.5 ul||2.0 ul||12.0 ul||20.0 ul
+
|6||1.4 ul||4.2 ul||1.0 ul||0.5 ul||2.9 ul||10.0 ul
 +
|-
 +
|7||1.4 ul||2.9 ul||1.0 ul||0.5 ul||4.2 ul||10.0 ul
 +
|-
 +
|8||1.4 ul||4.15 ul||1.0 ul||0.5 ul||2.95 ul||10.0 ul
 +
|-
 +
|9||1.4 ul||1.8 ul||1.0 ul||0.5 ul||5.3 ul||10.0 ul
 +
|-
 +
|10||1.4 ul||1.9 ul||1.0 ul||0.5 ul||5.2 ul||10.0 ul
 +
|-
 +
|12||1.4 ul||2.2 ul||1.0 ul||0.5 ul||4.9 ul||10.0 ul
 +
|-
 +
|13||1.4 ul||2.9 ul||1.0 ul||0.5 ul||4.2 ul||10.0 ul
 +
|-
 +
|15||1.4 ul||2.95 ul||1.0 ul||0.5 ul||4.15 ul||10.0 ul
 +
|-
 +
|17||1.4 ul||1.25 ul||1.0 ul||0.5 ul||5.75 ul||10.0 ul
 
|}
 
|}
  
−
<html>
+
Transformation was made.
−
<p>Vector:Insert</p>
+
−
    <p>3:1</p>
+
−
<p>RT 2h</p>
+
−
<p>Transformation at BL21.</p>
+
−
<p>CIP (+): No colonies were absorved.</p>
+
−
<p>CIP (-): Colony PCR will be made.</p>
+
  
−
</div>
+
<br>
  
−
    <a href="#" class="clickme" id="toggle" onclick="toggle_visibility('5');">Week 5<img src="https://static.igem.org/mediawiki/2014/f/f8/LMU14_arrow_down.png" id="5" class="tiger"></a>
 
  
−
<div class="box">
+
Colony PCR of “pTRE-1,13,15” (06.08.2015)
−
 
+
−
 
+
−
 
+
−
Creating “Plasmid pCAG” – Continue
+
−
</html>
+
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!Colony PCR from “pCAG” with “pTRE Luc fwd/SV40 polyA re. primers”
+
!Colony PCR  
 
|-
 
|-
−
|||MgCl₂||(NH₄)2SO₄||pTRE Luc fwd||SV40 rev||dNTP||Tag||ddH₂O||DNA||Total
+
|||MgCl₂||(NH₄)2SO₄||CMV fwd||SV40 rv||dNTP||Tag||ddH₂O||DNA||Total
 
|-
 
|-
 
|1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul
 
|1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul
 
|-
 
|-
−
|6X||15.0 ul||15.0 ul||6.0 ul||6.0 ul||3.0 ul||1.2 ul||73.8 ul||||120.0 ul
+
|20X||50.0 ul||50.0 ul||20.0 ul||20.0 ul||10.0 ul||2.0 ul||246.0 ul||||400.0 ul
 
|}
 
|}
−
 
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
 
!Cycling
 
!Cycling
 
|-
 
|-
−
|||95˚C||95˚C||55˚C||72˚C||72˚C||Cycle
+
|||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle
 
|-
 
|-
 
|Time||5’||30’’||30’’||1.5’||5’||35x
 
|Time||5’||30’’||30’’||1.5’||5’||35x
 
|}
 
|}
−
<html>
+
<a href="https://static.igem.org/mediawiki/2015/9/98/ATOMS-Turkiye_07_08_colony_pcr_1%2C2%2C3.jpeg" data-lightbox="image-1" class="img="left"><img src="https://static.igem.org/mediawiki/2015/9/98/ATOMS-Turkiye_07_08_colony_pcr_1%2C2%2C3.jpeg" width="410" height="360"></a>
−
<a href="https://static.igem.org/mediawiki/2015/4/40/ATOMS-Turkiye_pcag_cip_colony_pcr.jpeg" data-lightbox="image-1" class="img="center"><img src="https://static.igem.org/mediawiki/2015/4/40/ATOMS-Turkiye_pcag_cip_colony_pcr.jpeg" width="250" height="350"></a>
+
<p>Result: negative</p>
−
<p>Result: All bands were observed around 700 bp, results were negative. Ligation will be repeated.</p>
+
GEL GÖRÜNTÜSÜ
−
<p>(+) bant: 923 bp</p>
+
−
<p>(-) bant: 705 bp</p>
+
  
 
<br>
 
<br>
  
−
Creating “pCAG (Plasmid) – Repeat
+
Colony PCR of “5,6,7,9 – Gblocks” (06.08.2015)
−
</html>
+
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!PCR from “pCAGGS”
+
!Colony PCR  
 
|-
 
|-
−
|||dNTP||CAG fwd||CAG rev||Chicken β Akt. Rev||pCAGGS||Phusion Pol||Buffer||ddH₂O||Total
+
|||MgCl₂||(NH₄)2SO₄||VR||VF||dNTP||Tag||ddH₂O||DNA||Total
 
|-
 
|-
−
|1||2.0 ul||10.0 ul||10.0 ul||-||5.0 ul||1.0 ul||20.0 ul||52.0 ul||100.0 ul
+
|1x||2.5 ul||2.5 ul||0.5 ul||0.5 ul||0.5 ul||0.2 ul||13.3 ul||5.0 ul||25.0 ul
 
|-
 
|-
−
|2||2.0 ul||10.0 ul||-||10.0 ul||5.0 ul||1.0 ul||20.0 ul||52.0 ul||100.0 ul
+
|40X||100.0 ul||100.0 ul||20.0 ul||20.0 ul||20.0 ul||8.0 ul||532.0 ul||||800.0 ul
 
|}
 
|}
  
Line 606: Line 631:
 
!Cycling
 
!Cycling
 
|-
 
|-
−
|||98˚C||98˚C||64/68˚C||72˚C||72˚C||Cycle
+
|||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle
 
|-
 
|-
−
|Time||2’||10’’||30’’||1’||5’||35x
+
|Time||5’||30’’||30’’||1.5’||5’||35x
 
|}
 
|}
−
<html>
+
<p>Result: negative</p>
−
<a href="https://static.igem.org/mediawiki/2015/1/13/ATOMS-Turkiye_pcag_rwd_cag_rev.jpeg"  data-lightbox="image-1" class="img="center"><img src="https://static.igem.org/mediawiki/2015/1/13/ATOMS-Turkiye_pcag_rwd_cag_rev.jpeg" width="390" height="250"></a>
+
GEL GÖRÜNTÜSÜ
−
<p>Gel Extraction will made.</p>
+
  
 
<br>
 
<br>
  
−
</html>
+
Colony PCR of “7,8,10 – Gblocks” from Masterplate  (08.08.2015)
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
+
!Colony PCR
 
|-
 
|-
−
|1||eb||biospec||7/23/2015 6:56:38 PM||0.2||ng/ul||0.005||0.005||0.92||0.14||DNA||50.00
+
|||MgCl₂||(NH₄)2SO₄||T7 terminatör Rev.||T7 terminatör For.||dNTP||Tag||ddH₂O||DNA||Totalzz
 
|-
 
|-
−
|2||pcag e||biospec||7/23/2015 6:58:03 PM||42.9||ng/ul||0.858||0.450||1.91||1.97||DNA||50.00
+
|1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.5 ul||12.3 ul||5.0 ul||25.0 ul
 
|-
 
|-
−
|3||pcag y||biospec||7/23/2015 6:58:53 PM||23.3||ng/ul||0.466||0.233||2.00||1.94||DNA||50.00
+
|26X||65.0 ul||65.0 ul||26.0 ul||26.0 ul||13.0 ul||13.0 ul||319.8 ul||||527.8 ul
 
|}
 
|}
−
<html>
+
{| border="1" class="wikitable"
 +
!Cycling
 +
|-
 +
|||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle
 +
|-
 +
|Time||5’||30’’||30’’||1.5’||5’||35x
 +
|}
 +
<p>Result: negative</p>
 +
GEL GÖRÜNTÜSÜ
  
 
<br>
 
<br>
  
−
Resuspension of “Newly Arrived G-Blocks from IDT”
+
Digestion of “pET45” (09.08.2015)
−
<p>100 ul TE for all tubes.</p>
+
−
</html>
+
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!!!!!ng!!fmol!!TE ul!!fmol/ul!!ul of inserts for 75 fmol
+
!Digestion
 
|-
 
|-
−
|1||Toehold for cola||1000||1523||100||15.23||4.92449
+
|||pET45 (455ng/ul)||pSB1C3-RFP (400 ng/ul)||Cut Smart Buffer||XhoI (Neb)||BamHI-HF (Neb)||EcoRI-HF (Neb)||SpeI (Neb)||ddH₂O||Total
 
|-
 
|-
−
|2||TnrA-pTnrA-RFP||1000||1032||100||10.32||7.26744
+
|1||-||5.0 ul||2.0 ul||-||-||1.0 ul||1.0 ul||11.0 ul||20.0 ul
 
|-
 
|-
−
|3||ColA-KanR-dTer||1000||816||100||8.16||9.19118
+
|2||4.0 ul||-||2.0 ul||1.0 ul||1.0 ul||-||-||12.0 ul||20.0 ul
 +
|}
 +
<p>37˚C 3.5h</p>
 +
<p>Result: Positive. Gel extraction was made.</p>
 +
GEL GÖRÜNTÜSÜ
 +
 
 +
{| border="1" class="wikitable"
 +
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
 
|-
 
|-
−
|4||HNS for PET||500||1578||100||15.78||4.75285
+
|1||blank||biospec||8/9/2015 5:04:14 PM||-0.6||ng/ul||-0.011||0.005||3.41||0.68||DNA||50.00
 
|-
 
|-
−
|5||HNS-T108I for PET||500||1578||100||15.78||4.75285
+
|2||pet45 x+b||biospec||8/9/2015 5:05:26 PM||106.9||ng/ul||2.138||-0.017||0.59||0.45||DNA||50.00
 
|-
 
|-
−
|6||potB59-pomA for PET ||1000||892||100||8.92||8.40807
+
|4||psb1c3 e+s||biospec||8/9/2015 5:06:20 PM||133.3||ng/ul||2.666||0.913||1.90||0.40||DNA||50.00
 +
|}
 +
 
 +
 
 +
<html>
 +
</div>
 +
</section>
 +
 
 +
<section class="accordion">
 +
          <div>
 +
            <input id="august-week2" name="august-week2" type="checkbox" />
 +
            <label for="august-week2">Week 3, 10.16.-10.16.</label>
 +
            <article class="ac-small">
 +
</html>
 +
Ligation of “PET45 – HNS” (10.08.2015)
 +
{| border="1" class="wikitable"
 +
!Ligation
 
|-
 
|-
−
|7||Gad E – for PET||500||1291||100||12.91||5.80945
+
|||Vector||Insert||T4 DNA Ligase Buffer||T4 DNA Ligase||ddH₂O||Total
 
|-
 
|-
−
|8||TlpB for PET||1000||901||100||9.01||8.32408
+
|1x||2.1 ul||1.0 ul||2.0 ul||0.5 ul||14.4 ul||20.0 ul
 +
|}
 +
<p>16˚C 2h</p>
 +
 
 +
<br>
 +
 
 +
Digestion of “pSB1C3 – HNS”
 +
{| border="1" class="wikitable"
 +
!Ligation
 
|-
 
|-
−
|9||DAMP-Pex for PET/pcolA||1000||2089||100||20.89||3.59023
+
|||Gen||BamHI||XhoI||Cut Smart Buffer||ddH₂O||Total
 
|-
 
|-
−
|10||Tev Protease for PET||1000||1948||100||19.48||3.8501
+
|1x||11.5 ul||1.0 ul||1.0 ul||2.0 ul||4.5 ul||20.0 ul
 +
|}
 +
37˚C 3h
 +
 
 +
<br>
 +
 
 +
Colony PCR of “pTRE #13”
 +
{| border="1" class="wikitable"
 +
!Colony PCR
 
|-
 
|-
−
|11||miRNA switch- miR373- BS for pTET||500||2212||100||22.12||3.3906
+
|||MgCl₂||(NH₄)2SO₄||CMV for.||SV40 rev.||dNTP||Tag||ddH₂O||DNA||Total
 
|-
 
|-
−
|12||LacO- DsRed- miR26a-375 pC||1000||1709||100||17.09||4.38853
+
|1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul
 
|-
 
|-
−
|13||mLacI-miR373 BS for pTRE||1000||1280||100||12.8||5.85938
+
|10X||25.0 ul||25.0 ul||10.0 ul||10.0 ul||5.0 ul||2.0 ul||123.0 ul||||250.0 ul
 +
|}
 +
{| border="1" class="wikitable"
 +
!Cycling
 
|-
 
|-
−
|14||miRNA switch- miR 21 BS- miR 223||1000||1902||100||19.02||3.94322
+
|||95˚C||95˚C||60˚C||72˚C||72˚C||Cycle
 
|-
 
|-
−
|15||mLacI-miR223 BS miR 21 BS for pTRE||1000||1272||100||12.72||5.89623
+
|Time||5’||30’’||30’’||1.5’||5’||35x
 +
|}
 +
<p>Result: negative</p>
 +
GEL GÖRÜNTÜSÜ
 +
 
 +
<br>
 +
 
 +
Colony PCR of “PET45-4,5,6,7,8,9,10” (10.08.2015)
 +
{| border="1" class="wikitable"
 +
!Colony PCR
 
|-
 
|-
−
|16||Trigger RNA for pSB1C3||250||1910||100||19.1||3.9267
+
|||MgCl₂||(NH₄)2SO₄||T7 ter. fw||T7 ter. rev||dNTP||Tag||ddH₂O||DNA||Total
 
|-
 
|-
−
|17||PsicA for pSB1C3||1000||1546||100||15.46||4.86
+
|1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul
 
|-
 
|-
−
|18||MVF-sicA for ColA||1000||1042||100||10.42||7.20
+
|86X||215.0 ul||215.0 ul||86.0 ul||186.0 ul||43.0 ul||17.2 ul||1075.0 ul||||1720.0 ul
 
|}
 
|}
−
<html>
+
{| border="1" class="wikitable"
−
 
+
!Cycling
−
Not: 100ng pSB1C3 (2050 bp) ≈ 75 fmol
+
|-
 +
|||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle
 +
|-
 +
|Time||5’||30’’||30’’||1.5’||5’||35x
 +
|}
 +
<p>Result: Bands were at the expected section. Liquid Culture was made.</p>
 +
GEL GÖRÜNTÜLERI
  
 
<br>
 
<br>
  
−
PCR of “G-Blocks from IDT” (24.07.2015)
+
Digestion of „pColA-KanR  and „pSB1C3-Toehold“ (10.08.2015)
−
</html>
+
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!PCR from G-Bloks
+
!Digestion
 
|-
 
|-
−
|||MgCl₂||(NH₄)2SO₄||CMV fwd||SV40 rev||tetR rev||dNTP||Tag||ddH₂O||DNA||Total
+
|||pColA-KanR (500 ng/ul)||pSB1C3-Toehold (500 ng/ul)||NotI-HF (Neb)||Cut Smart Buffer||ddH₂O||Total||||||
 
|-
 
|-
−
|1x||2.5 ul||2.5 ul||0.5 ul||0.5 ul||0.5 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul
+
|1||11.6 ul||-||1.0 ul||2.0 ul||5.4 ul||20.0 ul||||||
 
|-
 
|-
−
|3x (pTEToff)||7.5 ul||7.5 ul||1.5 ul||-||1.5 ul||1.5 ul||1.6 ul||36.9 ul||||58.0 ul
+
|2||-||6.5 ul||1.0 ul||2.0 ul||10.5 ul||20.0 ul||||||
−
|-
+
−
|15x||37.5 ul||37.5 ul||7.5 ul||7.5 ul||-||7.5 ul||3.0 ul||184.5 ul||||285.0 ul
+
 
|}
 
|}
  
 +
<p>37˚C 2h</p>
 +
<p>pColA-KanR: 1.0 ul rSAP/37˚C 1h/-80˚C 20’’ heat shock</p>
 +
GEL GÖRÜNTÜSÜ
 +
 +
<br>
 +
 +
Ligation of “pColA-KanR and pSB1C3-Toehold”
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!Cycling for 3x
+
!Ligation!!
 
|-
 
|-
−
|||95˚C||95˚C||57˚C||72˚C||72˚C||Cycle
+
|||pColA-KanR (500 ng/ul)||pSb1C3-Toehold||T4 DNA Ligase Buffer (Thermo)||T4 DNA Ligase (Thermo)||ddH₂O||Total||Vector:Insert Ratios
 
|-
 
|-
−
|Time||5’||30’’||30’’||1.5’||5’||35x
+
|1||2.0 ul||1.0 ul||2.0 ul||1.0 ul||14.0 ul||20.0 ul||1:0.33
 +
|-
 +
|2||2.0 ul||3.0 ul||2.0 ul||1.0 ul||12.0 ul||20.0 ul||1:1
 +
|-
 +
|-K||2.0 ul||-||2.0 ul||1.0 ul||15.0 ul||20.0 ul||-
 
|}
 
|}
 +
<p>22˚C 1h</p>
  
 +
<br>
 +
 +
Mini Prep of “pColA-3,4,5”
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!Cycling for 15x
+
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
 
|-
 
|-
−
|||95˚C||95˚C||60˚C||72˚C||72˚C||Cycle
+
|1||blank||biospec||8/11/2015 11:11:42 PM||-0.2||ng/ul||-0.003||-0.007||0.49||0.13||DNA||50.00
 
|-
 
|-
−
|Time||5’||30’’||30’’||1.5’||5’||35x
+
|2||ColA-3||biospec||8/11/2015 11:13:07 PM||27.2||ng/ul||0.544||0.260||2.10||1.95||DNA||50.00
 +
|-
 +
|3||ColA-4||biospec||8/11/2015 11:14:38 PM||25.5||ng/ul||0.509||0.253||2.01||1.82||DNA||50.00
 +
|-
 +
|4||ColA-4(2)||biospec||8/11/2015 11:15:58 PM||25.8||ng/ul||0.517||0.255||2.022||1.81||DNA||50.00
 +
|-
 +
|5||ColA-5||biospec||8/11/2015 11:17:02 PM||44.4||ng/ul||0.887||0.449||1.98||1.76||DNA||50.00
 
|}
 
|}
−
<html>
+
<p>Result: The Band of pColA were at the expected section. 2000 pb</p>
−
<a href="https://static.igem.org/mediawiki/2015/8/8d/ATOMS-Turkiye_pcr_gblock.jpeg"  data-lightbox="image-1" class="img="center"><img src="https://static.igem.org/mediawiki/2015/8/8d/ATOMS-Turkiye_pcr_gblock.jpeg" width="390" height="250"></a>
+
(CUT CHECK GÖRÜNTÜSÜ)(12.08.2015)
−
<p>Results: Bands were at the expected section.</p>
+
  
 
<br>
 
<br>
  
−
Gel Extraction
+
Ligation of “#2 GBlock with pET45” (11.08.2015)
−
</html>
+
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
+
!Ligation
 
|-
 
|-
−
|1||blank||biospec||7/24/2015 12:40:09 PM||-0.8||ng/ul||-0.016||-0.020||0.79||0.24||DNA||50.00
+
|||pET45 (100 ng/ul)||#2 GBlock||T4 DNA Ligase Buffer ||T4 DNA Ligase ||ddH₂O||Total
 
|-
 
|-
−
|2||pSB1C3||biospec||7/24/2015 12:41:56 PM||42.2||ng/ul||0.848||0.430||1.97||2.05||DNA||50.00
+
|1x||1.0 ul||7.3 ul||2.0 ul||1.0 ul||8.7 ul||20.0 ul
 
|}
 
|}
−
<html>
+
<p>22˚C - 1’40’’</p>
 +
Transformation was made.
  
 
<br>
 
<br>
  
−
Ligation of „G-Blocks from IDT and pSB1C3“
+
Mini Prep
−
</html>
+
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!Ligation
+
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
 
|-
 
|-
−
|||Insert DNA||Vector (pSB1C3)||T4 DNA Buffer||T4 DNA Ligase||ddH₂O||Total
+
|1||blank||biospec||8/12/2015 7:38:28 PM||0.4||ng/ul||0.008||-0.001||-8.91||0.76||DNA||50.00
 
|-
 
|-
−
|1||4.9 ul||2.5 ul||2.0 ul||1.0 ul||9.8 ul||20.0 ul
+
|2||pet45 4-4||biospec||8/12/2015 7:40:21 PM||40.3||ng/ul||0.807||0.402||2.01||2.44||DNA||50.00
 
|-
 
|-
−
|2||7.2 ul||2.5 ul||2.0 ul||1.0 ul||7.3 ul||20.0 ul
+
|3||pet45 4-3||biospec||8/12/2015 7:41:10 PM||53.1||ng/ul||1.063||0.570||1.86||1.74||DNA||50.00
 
|-
 
|-
−
|3||9.2 ul||2.5 ul||2.0 ul||1.0 ul||5.3 ul||20.0 ul
+
|4||pet45 8-12||biospec||8/12/2015 7:41:55 PM||61.8||ng/ul||1.236||0.642||1.92||2.08||DNA||50.00
 
|-
 
|-
−
|4||4.8 ul||2.5 ul||2.0 ul||1.0 ul||9.7 ul||20.0 ul
+
|5||pet45 8-3||biospec||8/12/2015 7:42:37 PM||32.9||ng/ul||0.658||0.332||1.98||2.00||DNA||50.00
 
|-
 
|-
−
|5||4.8 ul||2.5 ul||2.0 ul||1.0 ul||9.7 ul||20.0 ul
+
|6||pet45 9-11||biospec||8/12/2015 7:44:03 PM||34.5||ng/ul||0.690||0.345||2.00||1.77||DNA||50.00
 
|-
 
|-
−
|6||8.4 ul||2.5 ul||2.0 ul||1.0 ul||6.1 ul||20.0 ul
+
|7||pet45 8-1||biospec||8/12/2015 7:44:54 PM||39.0||ng/ul||0.780||0.408||1.91||1.72||DNA||50.00
 
|-
 
|-
−
|7||5.8 ul||2.5 ul||2.0 ul||1.0 ul||8.7 ul||20.0 ul
+
|8||pet45 5-10||biospec||8/12/2015 7:45:49 PM||43.2||ng/ul||0.865||0.441||1.96||1.81||DNA||50.00
 
|-
 
|-
−
|8||4.9 ul||2.5 ul||2.0 ul||1.0 ul||9.6 ul||20.0 ul
+
|9||pet45 6-3||biospec||8/12/2015 7:46:42 PM||43.5||ng/ul||0.870||0.437||1.99||1.92||DNA||50.00
 +
|-
 +
|10||pet45 9-8||biospec||8/12/2015 7:47:23 PM||48.1||ng/ul||0.961||0.496||1.94||1.88||DNA||50.00
 +
|-
 +
|11||pet45 7-4||biospec||8/12/2015 7:48:06 PM||36.2||ng/ul||0.723||0.372||1.94||1.97||DNA||50.00
 +
|-
 +
|12||pet45 4-1||biospec||8/12/2015 7:48:50 PM||49.4||ng/ul||0.987||0.509||1.94||1.99||DNA||50.00
 +
|-
 +
|13||pet45 4-2||biospec||8/12/2015 7:49:43 PM||59.3||ng/ul||1.187||0.606||1.96||2.09||DNA||50.00
 +
|-
 +
|14||pet45 7-3||biospec||8/12/2015 7:50:33 PM||30.2||ng/ul||0.604||0.307||1.97||1.96||DNA||50.00
 +
|-
 +
|15||psb1c3 12||biospec||8/12/2015 7:51:18 PM||141.1||ng/ul||2.823||1.462||1.93||2.14||DNA||50.00
 +
|-
 +
|16||pet45 5-7||biospec||8/12/2015 7:52:04 PM||26.1||ng/ul||0.522||0.265||1.97||2.00||DNA||50.00
 +
|-
 +
|17||pet45 5-3||biospec||8/12/2015 7:52:49 PM||46.1||ng/ul||0.923||0.470||1.96||2.02||DNA||50.00
 +
|-
 +
|18||pet45 6-12||biospec||8/12/2015 7:53:36 PM||29.3||ng/ul||0.586||0.283||2.07||1.80||DNA||50.00
 +
|-
 +
|19||pet45 1-12||biospec||8/12/2015 7:54:25 PM||21.4||ng/ul||0.428||0.210||2.04||1.72||DNA||50.00
 +
|-
 +
|20||pet45 5-8||biospec||8/12/2015 7:55:08 PM||29.1||ng/ul||0.582||0.299||1.95||2.07||DNA||50.00
 +
|-
 +
|21||psb1c3 1||biospec||8/12/2015 7:55:51 PM||75.3||ng/ul||1.507||0.779||1.93||2.07||DNA||50.00
 +
|-
 +
|22||pet45 8-4||biospec||8/12/2015 7:56:33 PM||44.1||ng/ul||0.882||0.451||1.96||1.99||DNA||50.00
 +
|-
 +
|23||pet45 7-2||biospec||8/12/2015 7:57:16 PM||25.3||ng/ul||0.506||0.259||1.95||1.70||DNA||50.00
 
|}
 
|}
−
<html>
 
  
−
<p>RT 1h</p>
+
<br>
−
<p>Transformation was made.</p>
+
−
<p>(The results of psb1c3 gel extraction was lower. So we performed only the first 7 gene ligation.)</p>
+
  
−
</html>
 
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!1!!Toehold for cola
+
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
 
|-
 
|-
−
|2||TnrA-pTnrA-RFP
+
|1||blank||biospec||8/12/2015 7:17:01 PM||-0.3||ng/ul||-0.007||-0.015||0.47||0.23||DNA||50.00
 
|-
 
|-
−
|3||ColA-KanR-dTer
+
|2||psb1c3-4hns||biospec||8/12/2015 7:18:16 PM||61.9||ng/ul||1.237||0.652||1.90||2.17||DNA||50.00
 
|-
 
|-
−
|4||HNS for PET
+
|3||pet45 10-1||biospec||8/12/2015 7:19:32 PM||46.7||ng/ul||0.934||0.486||1.92||2.00||DNA||50.00
 
|-
 
|-
−
|5||HNS-T108I for PET
+
|4||pet45 10-6||biospec||8/12/2015 7:20:23 PM||52.0||ng/ul||1.040||0.549||1.89||1.88||DNA||50.00
 
|-
 
|-
−
|6||potB59-pomA for PET
+
|5||pet45 10-3||biospec||8/12/2015 7:21:14 PM||51.9||ng/ul||1.037||0.536||1.94||2.10||DNA||50.00
 
|-
 
|-
−
|7||Gad E – for PET
+
|6||pet45 9-6||biospec||8/12/2015 7:22:10 PM||37.7||ng/ul||0.755||0.389||1.94||1.99||DNA||50.00
 
|-
 
|-
−
|8||TlpB for cola (NEB1)
+
|7||pet45 10-8||biospec||8/12/2015 7:23:08 PM||85.3||ng/ul||1.705||1.002||1.70||0.93||DNA||50.00
 +
|-
 +
|8||psb1c3 16-trigger rna||biospec||8/12/2015 7:24:27 PM||125.7||ng/ul||2.514||1.401||1.79||1.32||DNA||50.00
 +
|-
 +
|9||pet45 10||biospec||8/12/2015 7:25:23 PM||84.7||ng/ul||1.694||1.025||1.65||0.76||DNA||50.00
 +
|-
 +
|10||pet45 6-6||biospec||8/12/2015 7:26:23 PM||100.3||ng/ul||2.006||1.124||1.79||1.24||DNA||50.00
 +
|-
 +
|11||pet45 8-2||biospec||8/12/2015 7:27:20 PM||67.5||ng/ul||1.349||0.778||1.73||0.91||DNA||50.00
 +
|-
 +
|12||pet45 7-1||biospec||8/12/2015 7:28:07 PM||52.5||ng/ul||1.050||0.549||1.91||1.83||DNA||50.00
 
|}
 
|}
−
<html>
 
  
 
<br>
 
<br>
  
−
Creating “Plasmid pCAG”
+
Cut Check of “pET45 1,4,5,6,7,8,9,10 – BamHI/XhoI” (13.08.2015)
−
</html>
+
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!Digestion
+
!Ligation
 
|-
 
|-
−
|||pTRE (1536 ng/ul)||pCAG (promoter) E||pCAG (promoter) Y||EcoRI||XhoI||Cut Smart Buffer||ddH₂O||Total||
+
|||Gen (250 ng/ul)||Cut Smart Buffer||BamHI (NEB)||XhoI (NEB)||ddH₂O||Total
 
|-
 
|-
−
|1||3.2 ul||-||-||0.5 ul||0.5 ul||2.0 ul||13.8 ul||20.0 ul||37˚C 2h
+
|1||11.7 ul||2.0 ul||1.0 ul||1.0 ul||4.3 ul||20.0 ul
 
|-
 
|-
−
|2||-||10 ul||-||0.5 ul||0.5 ul||2.0 ul||7.0 ul||20.0 ul||37˚C 2h
+
|4||6.2 ul||2.0 ul||1.0 ul||1.0 ul||9.8 ul||20.0 ul
 
|-
 
|-
−
|3||-||-||10 ul||0.5 ul||0.5 ul||2.0 ul||7.0 ul||20.0 ul||37˚C overnight
+
|5||8.6 ul||2.0 ul||1.0 ul||1.0 ul||7.4 ul||20.0 ul
 +
|-
 +
|6||8.6 ul||2.0 ul||1.0 ul||1.0 ul||7.4 ul||20.0 ul
 +
|-
 +
|7||8.3 ul||2.0 ul||1.0 ul||1.0 ul||7.7 ul||20.0 ul
 +
|-
 +
|8||7.4 ul||2.0 ul||1.0 ul||1.0 ul||8.6 ul||20.0 ul
 +
|-
 +
|9||6.7 ul||2.0 ul||1.0 ul||1.0 ul||9.3 ul||20.0 ul
 +
|-
 +
|10||5.9 ul||2.0 ul||1.0 ul||1.0 ul||10.1 ul||20.0 ul
 
|}
 
|}
−
<html>
+
<p>Dogru cikan koloniler BL21 bakterisine transformasyon edildi ve sivi kültür yapildi. Sonrasinda protein izolasyon ve WB yapilacak.</p>
−
<a href="https://static.igem.org/mediawiki/2015/f/f0/ATOMS-Turkiye_digastion_ptre_and_pcag.jpeg"  data-lightbox="image-1" class="img="center"><img src="https://static.igem.org/mediawiki/2015/f/f0/ATOMS-Turkiye_digastion_ptre_and_pcag.jpeg" width="250" height="390"></a>
+
 
−
<p>Bands were at the expected section. Gel extraction wil be made.</p>
+
GEL GÖRÜNTÜLERI
−
<p>The Final Concentration</p>
+
−
<p>pCAG E: 21.5 ng/ul</p>
+
−
pCAG Y: 12 ng/ul
+
  
 
<br>
 
<br>
  
−
</html>
 
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
 
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
 
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
 
|-
 
|-
−
|1||eb||biospec||7/25/2015 5:03:57 AM||1.4||ng/ul||0.028||0.006||4.60||0.48||DNA||50.00
+
|1||blank||biospec||8/13/2015 1:31:59 AM||0.0||ng/ul||-0.001||-0.005||0.17||0.08||DNA||50.00
 
|-
 
|-
−
|2||eb||biospec||7/25/2015 5:05:57 AM||0.0||ng/ul||0.000||-0.012||-0.03||0.06||DNA||50.00
+
|2||psb1c3-e+p||biospec||8/13/2015 1:32:58 AM||58.6||ng/ul||1.172||0.618||1.90||0.57||DNA||50.00
−
|-
+
−
|3||ptre delta tre ||biospec||7/25/2015 5:07:44 AM||194.0||ng/ul||3.880||2.033||1.91||2.20||DNA||50.00
+
 
|}
 
|}
−
<html>
 
  
 
<br>
 
<br>
  
−
Colony PCR of “pSB1C3 – GBlocks”
+
Colony PCR of “pTRE – 12,13 GBlocks” (14.08.2015)
−
</html>
+
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!PCR from “pCAGGS”
+
!Colony PCR  
 
|-
 
|-
−
|||PCR MM||VR fwd||VR rev||ddH₂O||Tag ||DNA||Total||||
+
|||MgCl₂||(NH₄)2SO₄||CMV fw||SV40 rv||dNTP||Tag||ddH₂O||DNA||Total
 
|-
 
|-
−
|1x||14.0 ul||1.0 ul||1.0 ul||7.5 ul||0.2 ul||5.0 ul||28.7 ul||||
+
|1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul
 
|-
 
|-
−
|33x||462.0 ul||33.0 ul||33.0 ul||247.5 ul||6.6 ul||165.0 ul||940.5 ul||||
+
|15X||37.5 ul||37.5 ul||15.0 ul||15.0 ul||7.5 ul||3.0 ul||184.5 ul||||300.0 ul
−
|-
+
−
|33x||462.0 ul||33.0 ul||33.0 ul||247.5 ul||6.6 ul||165.0 ul||940.5 ul||||
+
 
|}
 
|}
−
 
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
 
!Cycling
 
!Cycling
Line 834: Line 968:
 
|Time||5’||30’’||30’’||1.5’||5’||35x
 
|Time||5’||30’’||30’’||1.5’||5’||35x
 
|}
 
|}
−
<html>
+
<p>Sonuc: negative</p>
−
<a href="https://static.igem.org/mediawiki/2015/b/b1/ATOMS-Turkiye_25_07_colony_pcr.jpeg"  data-lightbox="image-1" class="img="center"><img src="https://static.igem.org/mediawiki/2015/b/b1/ATOMS-Turkiye_25_07_colony_pcr.jpeg" width="390" height="200"></a>
+
 
−
<a href="https://static.igem.org/mediawiki/2015/5/55/ATOMS-Turkiye_25_07_colony_pcr2.jpeg"  data-lightbox="image-1" class="img="center"><img src="https://static.igem.org/mediawiki/2015/5/55/ATOMS-Turkiye_25_07_colony_pcr2.jpeg" width="390" height="200"></a>
+
GEL GÖRÜNTÜSÜ
−
<a href="https://static.igem.org/mediawiki/2015/e/eb/ATOMS-Turkiye_25_07_colony_pcr3.jpeg"  data-lightbox="image-1" class="img="center"><img src="https://static.igem.org/mediawiki/2015/e/eb/ATOMS-Turkiye_25_07_colony_pcr3.jpeg" width="390" height="200"></a>
+
−
<p>Sonuc: 8-3 ve 4-9 bands were at the expected section. Liquid Culture was made.</p>
+
  
 
<br>
 
<br>
  
−
Colony PCR of “G-Blocks” (25.07.2015)
+
Colony PCR of “pColA – Toehold GFP” (14.08.2015)
−
</html>
+
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!Colony PCR of “2,3,6,8”
+
!Colony PCR  
 
|-
 
|-
−
|||MgCl₂||(NH₄)2SO₄||CMV fwd||SV40 rv||dNTP||Tag||ddH₂O||DNA||Total
+
|||MgCl₂||(NH₄)2SO₄||pColA fw||pColA rv||dNTP||Tag||ddH₂O||DNA||Total
 
|-
 
|-
−
|1x||2.5 ul||2.5 ul||0.5 ul||0.5 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||24.0 ul
+
|1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul
 
|-
 
|-
−
|6X||15.0 ul||15.0 ul||3.0 ul||3.0 ul||3.0 ul||1.2 ul||73.8 ul||||114.0 ul
+
|26X||65.0 ul||65.0 ul||26.0 ul||26.0 ul||13.0 ul||5.2 ul||319.8 ul||||520.0 ul
 
|}
 
|}
−
 
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!Colony PCR of “7,10,12,13”
+
!Cycling
 
|-
 
|-
−
|||MgCl₂||(NH₄)2SO₄||CMV fwd||SV40 rv||dNTP||Tag||ddH₂O||DNA||Total
+
|||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle
 
|-
 
|-
−
|1x||2.5 ul||2.5 ul||0.5 ul||0.5 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||24.0 ul
+
|Time||5’||30’’||30’’||1.5’||5’||35x
−
|-
+
−
|6X||15.0 ul||15.0 ul||3.0 ul||3.0 ul||3.0 ul||1.2 ul||73.8 ul||||114.0 ul
+
 
|}
 
|}
 +
<p>Result: 2-4, 2-8, 1-6 Bands were at the expected section.</p>
  
 +
<br>
 +
 +
Digestion of “pSB1C3-RFP” (14.08.2015)
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!Colony PCR of “1,4,9,15”
+
!Digestion
 
|-
 
|-
−
|||MgCl₂||(NH₄)2SO₄||CMV fwd||SV40 rv||dNTP||Tag||ddH₂O||DNA||Total
+
|||pSB1C3-RFP (400 ng/ul)||Cut Smart Buffer ||EcoRI-HF||PstI||ddH₂O||Total
 
|-
 
|-
−
|1x||2.5 ul||2.5 ul||0.5 ul||0.5 ul||0.5 ul||0.2 ul||13.3 ul||5.0 ul||25.0 ul
+
|1||5.0 ul||2.0 ul||1.0 ul||1.0 ul||11.0 ul||20.0 ul
 
|-
 
|-
−
|6X||15.0 ul||15.0 ul||3.0 ul||3.0 ul||3.0 ul||1.2 ul||79.8 ul||||120.0 ul
+
|2||5.0 ul||2.0 ul||1.0 ul||1.0 ul||11.0 ul||20.0 ul
 +
|-
 +
|3||5.0 ul||2.0 ul||1.0 ul||1.0 ul||11.0 ul||20.0 ul
 
|}
 
|}
 +
<p>37˚C – 3.5h</p>
 +
Gel extraction was made.
  
 +
<br>
 +
 +
Ligation of “pSB1C3-GBlocks”
 +
{| border="1" class="sortable"
 +
!Ligation
 +
|-
 +
|||Digested pSB1C3||GBlocks ||T4 DNA Ligase Buffer||T4 DNA Ligase||ddH₂O||Total
 +
|-
 +
|1||1.7 ul||2.0 ul||2.0 ul||1.0 ul||13.3 ul||20.0 ul
 +
|}
 +
<p>22˚C – 1.30h</p>
 +
Transformation with Neb10-β. Colony PCR will be made.
 +
 +
<br>
 +
 +
Colony PCR of “GBlocks – 2,5,6,7,8,9” (14.08.2015)
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!Colony PCR of “5,16”
+
!Colony PCR  
 
|-
 
|-
−
|||MgCl₂||(NH₄)2SO₄||CMV fwd||SV40 rv||dNTP||Tag||ddH₂O||DNA||Total
+
|||MgCl₂||(NH₄)2SO₄||VR||VF||dNTP||Tag||ddH₂O||DNA||Total
 
|-
 
|-
−
|1x||2.5 ul||2.5 ul||0.5 ul||0.5 ul||0.5 ul||0.2 ul||13.3 ul||5.0 ul||25.0 ul
+
|1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul
 
|-
 
|-
−
|2X||5.0 ul||5.0 ul||1.0 ul||1.0 ul||1.0 ul||0.4 ul||26.6 ul||||40.0 ul
+
|45X||112.5 ul||112.5 ul||45.0 ul||45.0 ul||22.5 ul||9.0 ul||553.5 ul||||900.0 ul
 +
|-
 +
|55X||137.5 ul||137.5 ul||55.0 ul||55.0 ul||27.5 ul||11.0 ul||676.5 ul||||1100.0 ul
 
|}
 
|}
−
 
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
 
!Cycling
 
!Cycling
 
|-
 
|-
−
|||95˚C||95˚C||60˚C||72˚C||72˚C||Cycle
+
|||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle
 
|-
 
|-
−
|Time||5’||30’’||2’||1.5’||5’||35x
+
|Time||5’||30’’||30’’||1.5’||5’||35x
 
|}
 
|}
−
<html>
+
GEL GÖRÜNTÜLERI
  
 
<br>
 
<br>
  
−
G-Blocks PCR / Gel Electrophoresis
+
Digestion/Gel Electrophoresis/Gel Extraction and Purification of “pSB1C3” (16.08.2015)
−
</html>
+
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!PCR
+
!Digestion
 
|-
 
|-
−
|||MgCl₂||(NH₄)2SO₄||CMV fwd||TetR rv||dNTP||Tag||ddH₂O||DNA||Total
+
|||DNA||FD||EcoRI||PstI||ddH₂O||Total
−
|-
+
−
|1x||2.5 ul||2.5 ul||0.5 ul||0.5 ul||0.5 ul||0.2 ul||13.3 ul||5.0 ul||25.0 ul
+
 
|-
 
|-
−
|6X||15.0 ul||15.0 ul||3.0 ul||3.0 ul||3.0 ul||1.2 ul||79.8 ul||||120.0 ul
+
|1x||2.5 ul||2.0 ul||1.0 ul||1.0 ul||13.5 ul||20.0 ul
 
|}
 
|}
 +
<p>37˚C 1h</p>
 +
 +
<br>
  
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!Cycling
+
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
 
|-
 
|-
−
|||95˚C||95˚C||60˚C||72˚C||72˚C||Cycle
+
|1||blank||biospec||8/16/2015 10:45:30 PM||0.1||ng/ul||0.003||-0.017||0.17||-0.14||DNA||50.00
 
|-
 
|-
−
|Time||5’||30’’||2’||1.5’||5’||35x
+
|2||psb1c3-e+p||biospec||8/16/2015 10:47:18 PM||14.0||ng/ul||0.279||0.145||1.92||0.15||DNA||50.00
 
|}
 
|}
  
−
<html>
+
<br>
−
<a href="https://static.igem.org/mediawiki/2015/b/b3/ATOMS-Turkiye_27_07_gblock_pcr.jpeg"  data-lightbox="image-1" class="img="center"><img src="https://static.igem.org/mediawiki/2015/b/b3/ATOMS-Turkiye_27_07_gblock_pcr.jpeg" width="390" height="230"></a>
+
−
<p>Result: negative</p>
+
−
 
+
−
</html>
+
  
 +
Colony PCR of “pSB1C3 7,10,12,13 and pET45 2” (14.08.2015)
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
+
!Colony PCR of “pSB1C3 7,10,12,13”
 
|-
 
|-
−
|1||blank||biospec||7/26/2015 7:04:13 PM||0.8||ng/ul||0.016||-0.014||-1.13||1.36||DNA||50.00
+
|||MgCl₂||(NH₄)2SO₄||VR||VF||dNTP||Tag||ddH₂O||DNA||Total
 
|-
 
|-
−
|2||blank||biospec||7/26/2015 7:05:51 PM||-0.4||ng/ul||-0.009||-0.017||0.52||0.17||DNA||50.00
+
|1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul
 
|-
 
|-
−
|3||colony pcr 8-3||biospec||7/26/2015 7:07:15 PM||87.9||ng/ul||1.758||0.908||1.94||2.09||DNA||50.00
+
|6X||15.0 ul||15.0 ul||6.0 ul||6.0 ul||3.0 ul||1.2 ul||73.8 ul||||120.0 ul
 +
|}
 +
{| border="1" class="wikitable"
 +
!Cycling
 
|-
 
|-
−
|4||colony pcr 4-9||biospec||7/26/2015 7:08:09 PM||101.4||ng/ul||2.027||1.089||1.86||1.74||DNA||50.00
+
|||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle
 
|-
 
|-
−
|5||colony pcr 4-9||biospec||7/26/2015 7:09:06 PM||71.4||ng/ul||1.429||0.749||1.91||1.96||DNA||50.00
+
|Time||5’||30’’||30’’||1.5’||5’||35x
−
|-
+
−
|6||colony pcr 8-3||biospec||7/26/2015 7:10:03 PM||87.9||ng/ul||1.758||0.910||1.93||2.10||DNA||50.00
+
−
|-
+
−
|7||colony pcr 4-9||biospec||7/26/2015 7:11:01 PM||57.1||ng/ul||1.142||0.596||1.91||1.77||DNA||50.00
+
−
|-
+
−
|8||colony pcr 4-9||biospec||7/26/2015 7:12:18 PM||77.2||ng/ul||1.543||0.820||1.88||1.76||DNA||50.00
+
 
|}
 
|}
 +
<p>Result: negative</p>
 +
GEL GÖRÜNTÜSÜ
  
−
<html>
 
 
<br>
 
<br>
  
−
Colony PCR of “pSB1C3- GBlocks”
 
−
</html>
 
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!Gradient PCR from pCAGGS
+
!Colony PCR of “pET45 2”
 
|-
 
|-
−
|||MgCl₂||(NH₄)2SO₄||VR fwd||VR rv||dNTP||ddH₂O||DNA||Total||
+
|||MgCl₂||(NH₄)2SO₄||VR||VF||dNTP||Tag||ddH₂O||DNA||Total
 
|-
 
|-
−
|1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||12.3 ul||5.0 ul||25.0 ul||
+
|1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul
 
|-
 
|-
−
|41X||102.5 ul||102.5 ul||41.0 ul||41.0 ul||20.5 ul||504.3 ul||||881.8 ul||
+
|12X||30.0 ul||30.0 ul||12.0 ul||12.0 ul||6.0 ul||2.4 ul||147.6 ul||||240.0 ul
 
|}
 
|}
−
 
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
 
!Cycling
 
!Cycling
Line 963: Line 1,108:
 
|Time||5’||30’’||30’’||1.5’||5’||35x
 
|Time||5’||30’’||30’’||1.5’||5’||35x
 
|}
 
|}
−
 
−
<html>
 
 
<p>Result: negative</p>
 
<p>Result: negative</p>
 +
GEL GÖRÜNTÜSÜ
  
 
<br>
 
<br>
  
−
Digestion of “pTEToff”
 
−
</html>
 
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!Digestion
+
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
 
|-
 
|-
−
|||pTEToff (1141 ng/ul)||SalI (Thermo)||HindIII (Thermo)||Fast Digest Buffer||ddH₂O||Total
+
|1||blank||biospec||8/16/2015 8:54:05 PM||-0.2||ng/ul||-0.004||-0.017||0.25||0.12||DNA||50.00
 
|-
 
|-
−
|Volume||3.1 ul||0.5 ul||0.5 ul||2.0 ul||13.9 ul||20.0 ul
+
|2||psb1c3 13-15||biospec||8/16/2015 8:55:29 PM||125.2||ng/ul||2.505||1.339||1.87||2.01||DNA||50.00
 +
|-
 +
|3||psb1c3 15-19||biospec||8/16/2015 8:56:24 PM||93.5||ng/ul||1.871||0.999||1.87||1.94||DNA||50.00
 +
|-
 +
|4||psb1c3 7-16||biospec||8/16/2015 8:57:33 PM||149.4||ng/ul||2.988||1.575||1.90||2.14||DNA||50.00
 +
|-
 +
|5||psb1c3 10-16||biospec||8/16/2015 8:58:18 PM||138.0||ng/ul||2.760||1.469||1.88||2.13||DNA||50.00
 +
|-
 +
|6||psb1c3 10-17||biospec||8/16/2015 8:59:03 PM||185.5||ng/ul||3.710||1.994||1.86||1.86||DNA||50.00
 +
|-
 +
|7||psb1c3 7-11||biospec||8/16/2015 8:00:54 PM||73.8||ng/ul||1.475||0.843||1.75||1.08||DNA||50.00
 +
|-
 +
|8||psb1c3 13-14||biospec||8/16/2015 8:01:53 PM||178.7||ng/ul||3.575||1.922||1.86||1.87||DNA||50.00
 +
|-
 +
|9||psb1c3 7-20||biospec||8/16/2015 8:02:54 PM||73.6||ng/ul||1.472||0.774||1.90||2.05||DNA||50.00
 +
|-
 +
|10||pcola toe 2-4||biospec||8/16/2015 8:03:57 PM||40.1||ng/ul||0.801||0.441||1.82||1.13||DNA||50.00
 +
|-
 +
|11||pcola toe 2-8||biospec||8/16/2015 8:05:31 PM||48.7||ng/ul||0.974||0.503||1.94||1.61||DNA||50.00
 +
|-
 +
|12||pcola toe 1-6||biospec||8/16/2015 8:06:09 PM||54.9||ng/ul||1.098||0.570||1.93||1.62||DNA||50.00
 +
|-
 +
|13||pcola blunt 1||biospec||8/16/2015 8:06:52 PM||23.9||ng/ul||0.479||0.244||1.96||1.24||DNA||50.00
 +
|-
 +
|14||pcola blunt 6||biospec||8/16/2015 8:07:30 PM||31.1||ng/ul||0.623||0.314||1.98||1.67||DNA||50.00
 
|}
 
|}
−
<html>
 
−
<p>37˚C 1h</p>
 
−
<a href="https://static.igem.org/mediawiki/2015/1/1f/ATOMS-Turkiye_06_07_ptetoff_digestion.jpeg"  data-lightbox="image-1" class="img="center"><img src="https://static.igem.org/mediawiki/2015/1/1f/ATOMS-Turkiye_06_07_ptetoff_digestion.jpeg" width="250" height="350"></a>
 
−
<p>Result: Bands were at the expected section. Gel purification was made.</p>
 
  
 
<br>
 
<br>
  
 +
 +
<html>
 +
</div>
 +
</section>
 +
 +
<section class="accordion">
 +
          <div>
 +
            <input id="august-week3" name="august-week3" type="checkbox" />
 +
            <label for="august-week3">Week 4, 17.08.-23.08.</label>
 +
            <article class="ac-small">
 
</html>
 
</html>
 +
Ligation of “pColA and pSB1C3 #9 (Damp-Pex)” (18.08.2015)
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
+
!Ligation
 
|-
 
|-
−
|1||blank||biospec||7/26/2015 4:18:56 PM||0.3||ng/ul||0.005||-0.010||-0.56||-0.38||DNA||50.00
+
|||pColA (Vector 50 ng/ul)||Insert (Damp-Pex)||T4 DNA Ligase Buffer||T4 DNA Ligase||ddH₂O||Total||Vector:Insert Ratio
 
|-
 
|-
−
|2||Ptetoff SalI hindIII||biospec||7/26/2015 4:20:23 PM||43.3||ng/ul||0.866||0.452||1.92||0.88||DNA||50.00
+
|9.1||2.0 ul||1.0 ul||2.0 ul||1.0 ul||14.0 ul||20.0 ul||1:0.33
 +
|-
 +
|9.2||2.0 ul||2.8 ul||2.0 ul||1.0 ul||12.2 ul||20.0 ul||1:1
 
|}
 
|}
  
−
<html>
 
 
<br>
 
<br>
  
−
Creating “Plasmid pCAG” – Continue  (27.07.2015)
+
Miniprep of “pTet-off DH5α /Top10/NEB5fα/NEB10β”
−
</html>
+
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!Ligation of „pTRE  TRE“ and „Digested pCAG“
+
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
 
|-
 
|-
−
|
+
|1||blank||biospec||8/18/2015 3:04:27 PM||0.4||ng/ul||0.008||-0.008||-1.01||1.01||DNA||50.00
 
|-
 
|-
−
|||pTRE  TRE (194 ng/ul)||pCAG E (21.5 ng/ul)||pCAG Y (12.0 ng/ul)||T4 DNA Ligase||Buffer||ddH₂O||Total
+
|2||top10||biospec||8/18/2015 3:05:19 PM||258.5||ng/ul||5.169||2.753||1.88||2.11||DNA||50.00
 
|-
 
|-
−
|1||1.0 ul||2.0 ul||-||0.5 ul||2.0 ul||14.5 ul||20.0 ul
+
|3||neb5a||biospec||8/18/2015 3:06:25 PM||30.6||ng/ul||0.612||0.324||1.89||1.34||DNA||50.00
 
|-
 
|-
−
|2||1.0 ul||-||3.6 ul||0.5 ul||2.0 ul||12.9 ul||20.0 ul
+
|4||neb5a||biospec||8/18/2015 3:07:30 PM||33.4||ng/ul||0.668||0.350||1.91||1.27||DNA||50.00
 +
|-
 +
|5||neb10beta||biospec||8/18/2015 3:08:22 PM||214.6||ng/ul||4.292||2.264||1.90||2.19||DNA||50.00
 
|}
 
|}
−
 
−
<html>
 
−
<p>Result: On the first plate was six colonies. On the second plate was nine colonies.</p>
 
−
Colony PCR will be made.
 
  
 
<br>
 
<br>
  
−
Colony PCR of „pCAG (plasmid)“
+
Colony PCR of “pET45 #2.8” (15.08.2015)
−
</html>
+
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!PCR  
+
!Colony PCR  
 
|-
 
|-
−
|||MgCl₂||(NH₄)2SO₄||pTRE Luc fwd||SV40 rv||dNTP||Tag||ddH₂O||DNA||Total
+
|||MgCl₂||(NH₄)2SO₄||VR||VF||dNTP||Tag||ddH₂O||DNA||Total
 
|-
 
|-
 
|1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul
 
|1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul
 
|-
 
|-
−
|16X||40.0 ul||40.0 ul||16.0 ul||16.0 ul||8.0 ul||3.2 ul||196.8 ul||||320.0 ul
+
|25X||62.5 ul||62.5 ul||25.0 ul||25.0 ul||12.5 ul||5.0 ul||307.5 ul||||500.0 ul
 
|}
 
|}
−
 
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
 
!Cycling
 
!Cycling
 
|-
 
|-
−
|||95˚C||95˚C||55˚C||72˚C||72˚C||Cycle
+
|||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle
 
|-
 
|-
−
|Time||5’||30’’||2’||1.5’||5’||35x
+
|Time||5’||30’’||30’’||1.5’||5’||35x
 
|}
 
|}
−
 
−
</html>
 
 
<p>Result: negative</p>
 
<p>Result: negative</p>
 
GEL GÖRÜNTÜSÜ
 
GEL GÖRÜNTÜSÜ
Line 1,043: Line 1,210:
 
<br>
 
<br>
  
−
Colony PCR of “pSB1C3 – Gblocks”
+
Colony PCR of “pSB1C3 17,6,8” (S. 118/15.08.2015)
−
<html>
+
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!PCR  
+
!Colony PCR of “pSB1C3 17,6,8”
 
|-
 
|-
−
|||MgCl₂||(NH₄)2SO₄||VR fwd||VR rv||dNTP||Tag||ddH₂O||DNA||Total
+
|||MgCl₂||(NH₄)2SO₄||VR||VF||dNTP||Tag||ddH₂O||DNA||Total
 
|-
 
|-
 
|1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul
 
|1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul
 
|-
 
|-
−
|57X||143.0 ul||143.0 ul||57.0 ul||57.0 ul||29.0 ul||11.4 ul||701.0 ul||||1141.4 ul
+
|32X||80.0 ul||80.0 ul||32.0 ul||32.0 ul||16.0 ul||6.4 ul||393.6 ul||||640.0 ul
−
|-
+
−
|57X||143.0 ul||143.0 ul||57.0 ul||57.0 ul||29.0 ul||11.4 ul||701.0 ul||||1141.4 ul
+
 
|}
 
|}
−
 
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
 
!Cycling
 
!Cycling
Line 1,064: Line 1,227:
 
|Time||5’||30’’||30’’||1.5’||5’||35x
 
|Time||5’||30’’||30’’||1.5’||5’||35x
 
|}
 
|}
−
 
+
<p>Result: 6: Liquid Culture was made.</p>
−
<html>
+
<p>8: sequencing will be done</p>
−
<a href="https://static.igem.org/mediawiki/2015/5/56/ATOMS-Turkiye_27%2C07%2Cpcr7.jpeg" data-lightbox="image-1" class="img="left"><img src="https://static.igem.org/mediawiki/2015/5/56/ATOMS-Turkiye_27%2C07%2Cpcr7.jpeg" width="410" height="360"></a>
+
<p>17: Colony PCR will be repeated.</p>
−
<a href="https://static.igem.org/mediawiki/2015/9/9a/ATOMS-Turkiye_27%2C07%2C15%2C16.jpeg" data-lightbox="image-1" class="img="left"><img src="https://static.igem.org/mediawiki/2015/9/9a/ATOMS-Turkiye_27%2C07%2C15%2C16.jpeg" width="410" height="360"></a>
+
GEL GÖRÜNTÜSÜ
−
<a href="https://static.igem.org/mediawiki/2015/b/bc/ATOMS-Turkiye_27%2C07%2Ccolony_pcr6.jpeg" data-lightbox="image-1" class="img="left"><img src="https://static.igem.org/mediawiki/2015/b/bc/ATOMS-Turkiye_27%2C07%2Ccolony_pcr6.jpeg" width="410" height="360"></a>
+
−
<a href="https://static.igem.org/mediawiki/2015/f/f0/ATOMS-Turkiye_27%2C08%2Claco%2Cdsred.jpeg" data-lightbox="image-1" class="https://static.igem.org/mediawiki/2015/f/f0/ATOMS-Turkiye_27%2C08%2Claco%2Cdsred.jpeg" width="410" height="360"></a>
+
−
<a href="https://static.igem.org/mediawiki/2015/8/8e/ATOMS-Turkiye_27%2C07%2Cpcr8.jpeg" data-lightbox="image-1" class="https://static.igem.org/mediawiki/2015/8/8e/ATOMS-Turkiye_27%2C07%2Cpcr8.jpeg" width="410" height="360"></a>
+
−
 
+
−
<p>Result: Only 16-9 is positive.</p>
+
−
 
+
  
 
<br>
 
<br>
  
−
Colony PCR of “pSB1C3 – Gblocks”
+
Colony PCR of “pColA blunt”
−
</html>
+
 
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!Colony PCR of “2,3,8,9,11,13,14”
+
!Colony PCR of “pColA blunt”
 
|-
 
|-
−
|||MgCl₂||(NH₄)2SO₄||VR fwd||VR rv||dNTP||Tag||ddH₂O||DNA||Total
+
|||MgCl₂||(NH₄)2SO₄||ColA fw||ColA rev||dNTP||Tag||ddH₂O||DNA||Total
 
|-
 
|-
 
|1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul
 
|1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul
 
|-
 
|-
−
|66X||165.0 ul||165.0 ul||66.0 ul||66.0 ul||33.0 ul||13.2 ul||811.8 ul||||1650.0 ul
+
|20X||50.0 ul||50.0 ul||20.0 ul||20.0 ul||10.0 ul||4.0 ul||246.0 ul||||400.0 ul
 
|}
 
|}
−
 
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
 
!Cycling
 
!Cycling
Line 1,096: Line 1,252:
 
|Time||5’||30’’||30’’||1.5’||5’||35x
 
|Time||5’||30’’||30’’||1.5’||5’||35x
 
|}
 
|}
−
 
−
<html>
 
−
<a href="https://static.igem.org/mediawiki/2015/a/a8/ATOMS-Turkiye_28%2C07%2Ccolony.jpeg" data-lightbox="image-1" class="img="left"><img src="https://static.igem.org/mediawiki/2015/a/a8/ATOMS-Turkiye_28%2C07%2Ccolony.jpeg" width="410" height="360"></a>
 
−
<a href="https://static.igem.org/mediawiki/2015/9/98/ATOMS-Turkiye_28%2C07%2C8%2C13%2C14.jpeg" data-lightbox="image-1" class="img="left"><img src="https://static.igem.org/mediawiki/2015/9/98/ATOMS-Turkiye_28%2C07%2C8%2C13%2C14.jpeg" width="410" height="360"></a>
 
−
 
 
<p>Result: negative</p>
 
<p>Result: negative</p>
−
 
+
GEL GÖRÜNTÜSÜ
  
 
<br>
 
<br>
  
−
Digestion of “HNS, HNS-T108I, potB59-pomA, GadE, TlpB, DAMP-Pex, Tev Protease” (27.07.2015)
+
PCR of “Q5 DNA Polymerase for pCAG” (19.08.2015)
−
<p>Inserts from GBlocks were digested to ligate with PET45.</p>
+
−
</html>
+
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!!!4!!5!!6!!7!!8!!9!!10
+
!PCR
 
|-
 
|-
−
|HNS (4)||10.0 ul||-||-||-||-||-||-
+
|||Template DNA||pCAG fw||pCAG rev||Master 2x Mix||ddH₂O||Total
 
|-
 
|-
−
|HNS-T108I (5)||-||10.0 ul||-||-||-||-||-
+
|1x||1.0 ul||5.0 ul||5.0 ul||25.0 ul||14.0 ul||50.0 ul
 +
|}
 +
{| border="1" class="wikitable"
 +
!Cycling
 
|-
 
|-
−
|potB59-pomA (6)||-||-||10.0 ul||-||-||-||-
+
|||98˚C||98˚C||70˚C||72˚C||72˚C||Cycle
 
|-
 
|-
−
|GadE (7)||-||-||-||10.0 ul||-||-||-
+
|Time||30’’||10’’||30’’||1’||5’||35x
 +
|}
 +
<p>Result: Bands were at the expected section. PCR clean up will be made.</p>
 +
GEL GÖRÜNTÜSÜ
 +
 
 +
<br>
 +
 
 +
Digestion of “pCAG – Clean up”
 +
{| border="1" class="wikitable"
 +
!Digestion
 
|-
 
|-
−
|TlpB (8)||-||-||-||-||10.0 ul||-||-
+
|||pCAG – Clean up (34 ng/ul)||pCAG – Clean up (61.6 ng/ul)||EcoRI-HF (NEB)||XhoI (NEB)||Cut Smart Buffer||ddH₂O||Total
 
|-
 
|-
−
|DAMP-Pex (9)||-||-||-||-||-||10.0 ul||-
+
|1||7.4 ul||-||0.5 ul||0.5 ul||2.0 ul||9.6 ul||20.0 ul
 
|-
 
|-
−
|Tev Protease (10)||-||-||-||-||-||-||10.0 ul
+
|2||-||4.1 ul||0.5 ul||0.5 ul||2.0 ul||12.9 ul||20.0 ul
 +
|}
 +
<p>37˚C overnight</p>
 +
 
 +
<br>
 +
 
 +
{| border="1" class="wikitable"
 +
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
 
|-
 
|-
−
|Cut Smart Buffer||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul
+
|1||blank||biospec||8/19/2015 7:08:39 PM||0.0||ng/ul||0.000||0.000||27.59||-0.07||DNA||50.00
 
|-
 
|-
−
|₂XhoI||0.5 ul||0.5 ul||0.5 ul||0.5 ul||0.5 ul||0.5 ul||0.5 ul
+
|2||pcag||biospec||8/19/2015 7:10:01 PM||61.6||ng/ul||1.233||0.686||1.80||1.02||DNA||50.00
 +
|}
 +
 
 +
<br>
 +
 
 +
Digestion of “pSB1C3 – mLacI miR373-BS” (19.08.2015)
 +
{| border="1" class="sortable"
 +
!Digestion
 
|-
 
|-
−
|BamHI-HF||0.5 ul||0.5 ul||0.5 ul||0.5 ul||0.5 ul||0.5 ul||0.5 ul
+
|||mLacI miR373-BS||EcoRI-HF (NEB)||XhoI (NEB)||Cut Smart Buffer||ddH₂O||Total||
 
|-
 
|-
−
|Milli-Q||7.0 ul||7.0 ul||7.0 ul||7.0 ul||7.0 ul||7.0 ul||7.0 ul
+
|1||3.4 ul||0.5 ul||0.5 ul||2.0 ul||13.9 ul||20.0 ul||
 
|}
 
|}
−
 
+
<p>37˚C 2.30h</p>
−
<html>
+
−
<p>Total: 20 ul</p>
+
−
<p>37˚C overnight</p>
+
−
Ligation will be made.
+
  
 
<br>
 
<br>
  
−
Ligation of “GBlocks (4,5,6,7,8,10)” and “PET45 (X+B)”
+
Ligation of “pSB1C3 – mLacI miR373-BS with pTRE”
−
</html>
+
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!!!4!!5!!6!!7!!8!!10
+
!Ligation
 
|-
 
|-
−
|Insert DNA||4.75 ul||4.75 ul||4.75 ul||4.75 ul||4.75 ul||4.75 ul
+
|||Digested mLacI miR373-BS with pTRE||pTRE||T4 DNA Ligase Buffer||T4 DNA Ligase||Cut Smart Buffer||ddH₂O||Total
 
|-
 
|-
−
|Vector (PET45)||2.20 ul||2.20 ul||.,20 ul||2.20 ul||2.20 ul||2.20 ul
+
|1||3.0 ul||1.0 ul||2.0 ul||1.0 ul||2.0 ul||13.0 ul||20.0 ul
 +
|}
 +
<p>22˚C 1.30h</p>
 +
<p>Transformation will be made.</p>
 +
 
 +
<br>
 +
 
 +
Colony PCR of “#6,8” (20.08.2015)
 +
{| border="1" class="wikitable"
 +
!Colony PCR
 
|-
 
|-
−
|Buffer||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul
+
|||MgCl₂||(NH₄)2SO₄||ColA fw||ColA rev||dNTP||Tag||ddH₂O||DNA||Total
 
|-
 
|-
−
|T4 DNA Ligase||0.5 ul||0.5 ul||0.5 ul||0.5 ul||0.5 ul||0.5 ul
+
|1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul
 
|-
 
|-
−
|ddH₂O||10.55 ul||10.55 ul||10.55 ul||10.55 ul||10.55 ul||10.55 ul
+
|25X||62.5 ul||62.5 ul||25.0 ul||25.0 ul||12.5 ul||5.0 ul||307.5 ul||||500.0 ul
 +
|}
 +
{| border="1" class="wikitable"
 +
!Cycling
 
|-
 
|-
−
|Total||20.0 ul||20.0 ul||20.0 ul||20.0 ul||20.0 ul||20.0 ul
+
|||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle
 +
|-
 +
|Time||5’||30’’||30’’||1.5’||5’||35x
 
|}
 
|}
−
 
+
<p>Result: Positive. Liquid Culture was made.</p>
−
<html>
+
−
Room Temperature 2h
+
  
 
<br>
 
<br>
  
−
</html>
 
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!!!11!!14
+
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
 
|-
 
|-
−
|Insert DNA||3.4 ul||3.95 ul
+
|1||blank||biospec||8/22/2015 12:11:34 PM||0.5||ng/ul||0.010||-0.003||-3.39||-3.90||DNA||50.00
 
|-
 
|-
−
|Vector (PET45)||2.20 ul||2.20 ul
+
|2||8-1||biospec||8/22/2015 12:12:38 PM||70.3||ng/ul||1.406||0.755||1.86||1.64||DNA||50.00
 
|-
 
|-
−
|Buffer||2.0 ul||2.0 ul
+
|3||8-3||biospec||8/22/2015 12:13:38 PM||80.7||ng/ul||1.613||0.846||1.91||2.11||DNA||50.00
 
|-
 
|-
−
|T4 DNA Ligase||0.5 ul||0.5 ul
+
|4||6-1||biospec||8/22/2015 12:14:32 PM||33.3||ng/ul||0.666||0.354||1.88||1.44||DNA||50.00
 +
|}
 +
 
 +
<br>
 +
 
 +
Cut Check of “#6,8”
 +
{| border="1" class="wikitable"
 +
!Cut Check
 
|-
 
|-
−
|ddH₂O||11.9 ul||11.35 ul
+
|||DNA (400 ng/ul)||EcoRI||PstI||Fast Digest Buffer||ddH₂O||Total
 
|-
 
|-
−
|Total||20.0 ul||20.0 ul
+
|1||5.7 ul||0.5 ul||0.5 ul||2.0 ul||11.3 ul||20.0 ul
 +
|-
 +
|2||4.8 ul||0.5 ul||0.5 ul||2.0 ul||12.2 ul||20.0 ul
 +
|-
 +
|3||12.0 ul||0.5 ul||0.5 ul||2.0 ul||5.0 ul||20.0 ul
 
|}
 
|}
 +
<p>37˚C 40’</p>
 +
<p>Result: positive</p>
 +
GEL GÖRÜNTÜSÜ
  
−
</html>
+
<br>
−
Room Temperature 2h
+
 
 +
Colony PCR “pSB1C3 17,6,8 – pColA, Damp-Pex, pTetoff” (19.08.2015)
 +
 
 +
GEL GÖRÜNTÜLERI
  
 
<br>
 
<br>
  
−
Creating of “pTRE – mLacI miRNA-BS” (27.07.2015)
+
Mini Prep of “pSB1C3-#17”
−
</html>
+
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!Digestion of “pTRE” and G-Blocks”
+
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
 +
|-
 +
|1||blank||biospec||8/20/2015 5:04:31 PM||0.6||ng/ul||0.013||0.007||1.93||1.40||DNA||50.00
 
|-
 
|-
−
|||pTRE ||EcoRI-HF||BamHI-HF||mLacI-miR 373 BS||mLacI-miR (21-223) BS||Cut Smart||ddH₂O||Total||
+
|2||psb1c3 17-2||biospec||8/20/2015 5:05:28 PM||78.9||ng/ul||1.579||0.821||1.92||2.17||DNA||50.00
 
|-
 
|-
−
|1||3.1 ul||0.5 ul||0.5 ul||-||-||2.0 ul||13.9 ul||20.0 ul||37˚C-3h Gel Elect.
+
|3||psb1c3 17-5||biospec||8/20/2015 5:06:18 PM||77.5||ng/ul||1.551||0.806||1.92||2.08||DNA||50.00
 
|-
 
|-
−
|2||-||0.5 ul||0.5 ul||10.0 ul||-||2.0 ul||7.0 ul||20.0 ul||37˚C overnight/80˚C-10’ heat inactivation
+
|4||psb1c3 17-8||biospec||8/20/2015 5:07:02 PM||87.5||ng/ul||1.749||0.910||1.92||2.08||DNA||50.00
 
|-
 
|-
−
|3||-||0.5 ul||0.5 ul||-||10.0 ul||2.0 ul||7.0 ul||20.0 ul||37˚C overnight/80˚C-10’ heat inactivation
+
|5||psb1c3 17-10||biospec||8/20/2015 5:07:47 PM||105.4||ng/ul||2.108||1.087||1.94||2.15||DNA||50.00
 
|}
 
|}
  
−
<html>
+
<br>
−
<p>Gel purification was made.</p>
+
 
−
GEL GÖRÜNTÜSÜ
+
Digestion of “pSB1C3 T7-10 and pET45 #4-1, #5-3, #6-6, #8-15” (21.08.2015)
 +
{| border="1" class="wikitable"
 +
!Digestion pET45 #4-1, #5-3, #6-6, #8-15
 +
|-
 +
|||DNA||BamHI-HF (NEB)||XhoI (NEB)||Cut Smart Buffer||ddH₂O||Total||
 +
|-
 +
|#4-1||10.1 ul||1.0 ul||1.0 ul||2.0 ul||5.9 ul||20.0 ul||
 +
|-
 +
|#5-3||10.8 ul||1.0 ul||1.0 ul||2.0 ul||5.2 ul||20.0 ul||
 +
|-
 +
|#6-6||5.0 ul||1.0 ul||1.0 ul||2.0 ul||11.0 ul||20.0 ul||
 +
|-
 +
|#8-15||16.0 ul||1.0 ul||1.0 ul||2.0 ul||-||20.0 ul||
 +
|}
 +
<p>37˚C 3h</p>
  
 
<br>
 
<br>
  
−
</html>
 
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
+
!Digestion pSB1C3 T7-10
 
|-
 
|-
−
|1||blank||biospec||7/27/2015 2:26:05 PM||0.3||ng/ul||0.007||-0.004||-1.71||0.20||DNA||50.00
+
|||DNA||BamHI-HF (NEB)||XhoI (NEB)||Cut Smart Buffer||ddH₂O||Total||
 
|-
 
|-
−
|2||pTRE e+b||biospec||7/27/2015 2:27:08 PM||92.9||ng/ul||1.859||1.015||1.83||1.24||DNA||50.00
+
|#4-1||4.8 ul||1.0 ul||1.0 ul||2.0 ul||11.2 ul||20.0 ul||
 
|}
 
|}
 +
<p>1ul rSAP / 37˚C 1h</p>
  
−
<html>
 
 
<br>
 
<br>
  
−
Creating of “pTRE – mLacI miRBS” (27.07.2015)
 
−
</html>
 
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!Ligation of „pTRE” and “mLacI miRBS”
+
!Ligation
 
|-
 
|-
−
|||Digested pTRE||D. mLacI miR373 BS||D. mLacI miR(21-223) BS||T4 DNA Ligase||T4 DNA Buffer||ddH₂O||Total
+
|||Vector (pSB1C3)||Insert||T4 DNA Ligase Buffer||T4 DNA Ligase||ddH₂O||Total||
 
|-
 
|-
−
|1||1.0 ul||5.9 ul||-||2.0 ul||0.5 ul||10.6 ul||20.0 ul
+
|#4||1.0 ul||1.0 ul||2.0 ul||1.0 ul||15.0 ul||20.0 ul||
 
|-
 
|-
−
|2||1.0 ul||-||5.9 ul||2.0 ul||0.5 ul||10.6 ul||20.0 ul
+
|#5||1.0 ul||1.0 ul||2.0 ul||1.0 ul||15.0 ul||20.0 ul||
 +
|-
 +
|#6||1.0 ul||1.0 ul||2.0 ul||1.0 ul||15.0 ul||20.0 ul||
 +
|-
 +
|#8||1.0 ul||1.0 ul||2.0 ul||1.0 ul||15.0 ul||20.0 ul||
 
|}
 
|}
−
<html>
+
<p>22˚C 2h</p>
−
<p>Room Temperature 2h</p>
+
<p>-80 ˚C 20’’ Heat Inaktivation</p>
−
Transformation was made with TOP10.
+
  
 
<br>
 
<br>
  
−
Creating of “pTEToff – miRBS” (27.07.2015)
+
Colony PCR of “pTRE - #13 mLacI-miR373 BS” (21.08.2015)
−
</html>
+
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!Digestion
+
!Colony PCR
 
|-
 
|-
−
|||pTEToff (2.5 ng/ul)||miRNA switch miR373 BS||miRNA switch miR(223-21) BS||SalI (FD)||HindIII (FD)||Fast Digest Buffer||ddH₂O||Total||
+
|||MgCl₂||(NH₄)2SO₄||CMV fw||SV40 rv||dNTP||Tag||ddH₂O||DNA||Total
 
|-
 
|-
−
|1||2.0 ul||-||-||0.5 ul||0.5 ul||5.0 ul||42.0 ul||50.0 ul||37˚C-3h Gel Elect.
+
|1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul
 
|-
 
|-
−
|2||-||10.0 ul||-||0.5 ul||0.5 ul||2.0 ul||7.0 ul||20.0 ul||37˚C overnight/80˚C-10’ heat inactivation
+
|22X||55.0 ul||55.0 ul||22.0 ul||22.0 ul||11.0 ul||4.4 ul||270.6 ul||||440.0 ul
 +
|}
 +
{| border="1" class="wikitable"
 +
!Cycling
 
|-
 
|-
−
|3||-||-||10.0 ul||0.5 ul||0.5 ul||2.0 ul||7.0 ul||20.0 ul||37˚C overnight/80˚C-10’ heat inactivation
+
|||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle
 +
|-
 +
|Time||5’||30’’||30’’||1.5’||5’||35x
 
|}
 
|}
 +
<p>Results: negative</p>
 +
GEL GÖRÜNTÜLERI
  
−
<html>
 
 
<br>
 
<br>
  
−
</html>
+
Mini Prep of “pSB1C3 #6-25,26”
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
 
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
 
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
 
|-
 
|-
−
|1||blank||biospec||7/27/2015 8:35:05 PM||0.0||ng/ul||0.000||-0.013||-0.01||0.01||DNA||50.00
+
|1||blank||biospec||8/20/2015 10:01:33 AM||0.4||ng/ul||0.007||-0.003||-2.12||-1.13||DNA||50.00
 
|-
 
|-
−
|2||pTRE s+h||biospec||7/27/2015 8:36:16 PM||76.7||ng/ul||1.534||0.810||1.89||1.05||DNA||50.00
+
|2||psb1c3 #6-25||biospec||8/20/2015 10:02:45 AM||41.0||ng/ul||0.820||0.421||1.95||1.93||DNA||50.00
 +
|-
 +
|3||psb1c3 #6-26||biospec||8/20/2015 10:03:50 AM||46.0||ng/ul||0.920||0.464||1.98||1.97||DNA||50.00
 
|}
 
|}
  
−
<html>
 
 
<br>
 
<br>
  
−
Creating of “pTEToff – miRNA Switch miR BS” (28.07.2015)
+
Cut Check of “pSB1C3 #6-25,26”
−
</html>
+
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!Ligation of „pTRE” and “mLacI miRBS”
+
!Cut Check
 
|-
 
|-
−
|||Digested pTEToff||D. mLacI miR373 BS||D. mLacI miR(21-223) BS||T4 DNA Ligase||T4 DNA Buffer||ddH₂O||Total
+
|||DNA ||EcoRI-HF||PstI-HF||Cut Smart Buffer||ddH₂O||Total
 
|-
 
|-
−
|1||2.2 ul||3.4 ul||-||2.0 ul||0.5 ul||10.9 ul||20.0 ul
+
|#6-25||6.1 ul||0.5 ul||0.5 ul||2.0 ul||10.9 ul||20.0 ul
 
|-
 
|-
−
|2||2.2 ul||-||4.0 ul||2.0 ul||0.5 ul||11.3 ul||20.0 ul
+
|#6-26||5.5 ul||0.5 ul||0.5 ul||2.0 ul||11.5 ul||20.0 ul
 
|}
 
|}
−
<html>
+
<p>37˚C 3h</p>
−
<p>Room Tempretare 2h</p>
+
<p>Result: negative</p>
−
Transformation was made.
+
  
 
<br>
 
<br>
  
−
Creating of “pCAG”
+
Digestion of “pTRE #12”
−
</html>
+
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!Digestion of “pCAG (Promotor)”
+
!Digestion
 
|-
 
|-
−
|||pCAG (promoter) E||pCAG (promoter) Y||EcoRI-HF||XhoI-HF||Cut Smart Buffer||ddH₂O||Total||||
+
|||pTRE #12 (5000 ng/ul)||EcoRI-HF||XhoI (NEB)||Cut Smart Buffer||ddH₂O||Total
 
|-
 
|-
−
|1||10.0 ul||-||0.5 ul||0.5 ul||2.0 ul||7.0 ul||20.0 ul||37˚C overnight||
+
|1x||16.0 ul||0.5 ul||0.5 ul||2.0 ul||1.0 ul||20.0 ul
−
|-
+
−
|2||-||10.0 ul||0.5 ul||0.5 ul||2.0 ul||7.0 ul||20.0 ul||37˚C overnight||
+
 
|}
 
|}
 +
<p>37˚C 3h</p>
 +
<p>Result: negative</p>
 +
GEL GÖRÜNTÜSÜ
  
−
<html>
 
 
<br>
 
<br>
  
−
Mini Prep of “Colony PCR 16-9”
+
Digestion of “Damp-Pex pSB1C3” (22.08.2015)
−
<html>
+
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
+
!Digestion pSB1C3 T7-10
 
|-
 
|-
−
|1||blank||biospec||7/28/2015 12:11:17 PM||-1.9||ng/ul||-0.038||-0.049||0.77||0.19||DNA||50.00
+
|||DNA (500 ng/ul)||NotI||Cut Smart Buffer||ddH₂O||Total||||
 
|-
 
|-
−
|2||blank||biospec||7/28/2015 12:12:22 PM||-0.4||ng/ul||-0.007||-0.014||0.53||0.30||DNA||50.00
+
|#9-5||3.42 ul||1.0 ul||2.0 ul||13.58 ul||20.0 ul||||
 
|-
 
|-
−
|3||colony pcr 16-9||biospec||7/28/2015 12:13:33 PM||84.9||ng/ul||1.698||0.881||1.93||2.02||DNA||50.00
+
|#9-10||3.56 ul||1.0 ul||2.0 ul||13.44 ul||20.0 ul||||
−
|-
+
−
|4||colony pcr 16-9||biospec||7/28/2015 12:14:24 PM||76.3||ng/ul||1.527||0.797||1.92||1.95||DNA||50.00
+
 
|}
 
|}
 +
<p>37˚C 3h</p>
  
−
<html>
 
 
<br>
 
<br>
  
−
Mini Prep of “pTEToff”
+
Ligation of “Damp-Pex pColA”
−
</html>
+
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
+
!Ligation
 
|-
 
|-
−
|1||blank||biospec||7/30/2015 4:44:01 PM||0.4||ng/ul||0.008||-0.005||-1.55||1.11||DNA||50.00
+
|||pColA (100 ng/ul)||Insert (36 ng/ul)||T4 DNA Ligase Buffer||T4 DNA Ligase||ddH₂O||Total||
 
|-
 
|-
−
|2||ptetoff neb||biospec||7/30/2015 4:45:00 PM||442.2||ng/ul||8.884||4.683||1.89||2.20||DNA||50.00
+
|1x||2.8 ul||5.41 ul||2.0 ul||1.0 ul||15.0 ul||20.0 ul||
−
|-
+
−
|3||ptetoff bl21||biospec||7/30/2015 4:46:08 PM||314.3||ng/ul||6.286||3.317||1.90||2.22||DNA||50.00
+
 
|}
 
|}
 +
<p>22˚C 1.30h</p>
 +
<p>Transformation with NEB5α.</p>
  
−
<html>
 
 
<br>
 
<br>
  
−
Cut Check of “HNS toehold and Trigger RNA” (30.07.2015)
+
Colony PCR of “Damp-Pex + pColA” (23.08.2015)
−
<html>
+
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!!!HNS (70 ng/ul)!!TlpB (87 ng/ul)!!Trigger RNA (80 ng/ul)!!EcoRI (Thermo)!!PstI (Thermo)!!Fast Digest Buffer!!ddH₂O!!Total!!
+
!Colony PCR
 
|-
 
|-
−
|1||3.5 ul||-||-||1.0 ul||1.0 ul||2.0 ul||12.5 ul||20.0 ul||37˚C 20min
+
|||MgCl₂||(NH₄)2SO₄||pColA fw||pColA rv||dNTP||Tag||ddH₂O||DNA||Total
 
|-
 
|-
−
|2||-||3.0 ul||-||1.0 ul||1.0 ul||2.0 ul||13.0 ul||20.0 ul||37˚C 20min
+
|1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul
 
|-
 
|-
−
|3||-||-||3.0 ul||1.0 ul||1.0 ul||2.0 ul||13.0 ul||20.0 ul||37˚C 20min
+
|26X||65.0 ul||65.0 ul||26.0 ul||26.0 ul||13.0 ul||5.2 ul||319.8 ul||||520.0 ul
 
|}
 
|}
−
 
+
{| border="1" class="wikitable"
−
<html>
+
!Cycling
−
<p>Gel Electropheres was made.</p>
+
|-
−
 
+
|||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle
−
<p>Result: Bands were at the expected section.</p>
+
|-
−
<p>HNS: 480 bp</p>
+
|Time||5’||30’’||30’’||1.5’||5’||35x
−
<p>tgRNA:˞ 100bp</p>
+
|}
−
<p>Toehold: ˃1000bp</p>
+
<p>Result: #5-4, #5-9 Liquid Culture was made.</p>
−
<a href="https://static.igem.org/mediawiki/2015/7/7e/ATOMS-Turkiye_30%2C06%2Ccutcheck.jpeg" data-lightbox="image-1" class="img="left"><img src="https://static.igem.org/mediawiki/2015/7/7e/ATOMS-Turkiye_30%2C06%2Ccutcheck.jpeg" width="410" height="360"></a>
+
GEL GÖRÜNTÜSÜ
  
 
<br>
 
<br>
  
−
Colony PCR of „9-10 GBlocks“
+
Mini Prep of “pTetoff #14-4,5,8” (22.08.2015)
−
<a href="https://static.igem.org/mediawiki/2015/1/1f/ATOMS-Turkiye_31%2C07%2C9%2Ccolony_pcr.jpeg" data-lightbox="image-1" class="img="left"><img src="https://static.igem.org/mediawiki/2015/1/1f/ATOMS-Turkiye_31%2C07%2C9%2Ccolony_pcr.jpeg" width="410" height="360"></a>
+
{| border="1" class="wikitable"
−
<a href="https://static.igem.org/mediawiki/2015/9/92/ATOMS-Turkiye_31%2C07%2C10%2Ccolony.jpeg" data-lightbox="image-1" class="img="left"><img src="https://static.igem.org/mediawiki/2015/9/92/ATOMS-Turkiye_31%2C07%2C10%2Ccolony.jpeg" width="410" height="360"></a>
+
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
−
<p>Result: negative</p>
+
|-
−
<p>Expected: ˞1100 bp</p>
+
|1||blank||biospec||8/22/2015 11:01:09 AM||0.1||ng/ul||0.002||-0.008||-0.23||-0.10||DNA||50.00
−
<p>Observed: ˞300 bp</p>
+
|-
 +
|2||ptetoff 14-4||biospec||8/22/2015 11:02:05 AM||159.6||ng/ul||3.193||1.678||1.90||2.00||DNA||50.00
 +
|-
 +
|3||ptetoff 14-5||biospec||8/22/2015 11:03:21 AM||141.1||ng/ul||2.822||1.466||1.93||1.97||DNA||50.00
 +
|-
 +
|4||ptetoff 14-8||biospec||8/22/2015 11:04:14 AM||367.5||ng/ul||7.350||3.969||1.85||2.11||DNA||50.00
 +
|}
  
 
<br>
 
<br>
  
−
Gel extraction 1-6
 
−
</html>
 
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
+
!Cut Check I
 
|-
 
|-
−
|1||blank||biospec||8/2/2015 12:01:41 AM||0.2||ng/ul||0.004||-0.002||-2.02||17.14||DNA||50.00
+
|||DNA (250 ng/ul)||SalI (FD)||HindIII (FD)||Fast Digest Buffer||ddH₂O||Total
 
|-
 
|-
−
|2||psb1c3||biospec||8/2/2015 12:31:14 AM||43.0||ng/ul||0.861||0.462||1.87||0.94||DNA||50.00
+
|#14-4||1.6 ul||0.5 ul||0.5 ul||2.0 ul||15.4 ul||20.0 ul
 +
|-
 +
|#14-5||1.8 ul||0.5 ul||0.5 ul||2.0 ul||15.2 ul||20.0 ul
 +
|-
 +
|#14-8||0.7 ul||0.5 ul||0.5 ul||2.0 ul||16.3 ul||20.0 ul
 
|}
 
|}
 +
<p>37˚C 2h</p>
 +
<p>Result: negative</p>
 +
 +
GEL GÖRÜNTÜSÜ
  
−
<html>
 
 
<br>
 
<br>
  
−
Gel extraction 8-9
 
−
</html>
 
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
+
!Cut Check II
 
|-
 
|-
−
|1||blank||biospec||8/2/2015 12:27:36 AM||-0.4||ng/ul||-0.009||-0.012||0.74||-0.09||DNA||50.00
+
|||DNA (250 ng/ul)||SalI (FD)||NotI (FD)||Fast Digest Buffer||ddH₂O||Total
 
|-
 
|-
−
|2||psb1c3 atoms||biospec||8/2/2015 12:34:11 AM||10.4||ng/ul||0.208||0.111||1.87||0.03||DNA||50.00
+
|#14-4||1.6 ul||0.5 ul||0.5 ul||2.0 ul||15.4 ul||20.0 ul
 +
|-
 +
|#14-5||1.8 ul||0.5 ul||0.5 ul||2.0 ul||15.2 ul||20.0 ul
 +
|-
 +
|#14-8||0.7 ul||0.5 ul||0.5 ul||2.0 ul||16.3 ul||20.0 ul
 
|}
 
|}
 +
<p>37˚C 30’’</p>
 +
<p>Result: negative</p>
 +
GEL GÖRÜNTÜSÜ
  
−
<html>
 
 
<br>
 
<br>
  
−
Colony PCR of „pSB1C3 – Gblocks“
+
Colony PCR of “pSB1C3 – T7 #4,5,6,8” (23.08.2015)
−
</html>
+
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
 
!Colony PCR  
 
!Colony PCR  
 
|-
 
|-
−
|||MgCl₂||(NH₄)2SO₄||VR fwd||VR rv||dNTP||Tag||ddH₂O||DNA||Total
+
|||MgCl₂||(NH₄)2SO₄||pTRE Luc fw||SV40 rv||dNTP||Tag||ddH₂O||DNA||Total
 
|-
 
|-
−
|1x||2.5 ul||2.5 ul||0.5 ul||0.5 ul||0.5 ul||0.2 ul||13.3 ul||5.0 ul||25.0 ul
+
|1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul
 
|-
 
|-
−
|17X||42.5 ul||42.5 ul||8.5 ul||8.5 ul||8.5 ul||3.4 ul||226.1 ul||||340.0 ul
+
|50x||125.0 ul||125.0 ul||50.0 ul||50.0 ul||25.0 ul||10.0 ul||615.0 ul||||1000.0 ul
 
|}
 
|}
−
 
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
 
!Cycling
 
!Cycling
Line 1,406: Line 1,629:
 
|Time||5’||30’’||30’’||1.5’||5’||35x
 
|Time||5’||30’’||30’’||1.5’||5’||35x
 
|}
 
|}
 +
GEL GÖRÜNTÜSÜ
 +
</div>
 +
</section>
 +
      <section class="accordion">
 +
          <div>
 +
            <input id"august-week4" name="august-week4" type="checkbox" />
 +
            <label for="august-week4">Week 1, 24.08.-31.08.</label>
 +
            <article class="ac-small">
 +
</html>
 +
Digestion of “pTRE #12-2” (24.08.2015)
 +
{| border="1" class="wikitable"
 +
!Digestion
 +
|-
 +
|||pTRE #12-2 (2000 ng/ul)||EcoRI-HF||XhoI-NEB||Cut Smart Buffer||ddH₂O||Total||
 +
|-
 +
|1x||6.4 ul||0.5 ul||0.5 ul||2.0 ul||10.6 ul||20.0 ul||
 +
|}
 +
<p>37˚C 2h</p>
 +
<p>Result: Bands were at the expected section. Gel Extraction was made.</p>
  
−
<html>
+
<br>
−
<p>Result: negative</p>
+
−
GEL GÖRÜNTÜLERI
+
  
 +
Mini Prep
 +
{| border="1" class="wikitable"
 +
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
 +
|-
 +
|1||blank||biospec||8/24/2015 1:28:49 AM||0.0||ng/ul||0.001||-0.013||-0.07||-0.07||DNA||50.00
 +
|-
 +
|2||ptre gel||biospec||8/24/2015 1:30:29 AM||15.3||ng/ul||0.306||0.164||1.87||0.65||DNA||50.00
 +
|}
  
−
</div>
+
<br>
  
 +
{| border="1" class="wikitable"
 +
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
 +
|-
 +
|1||blank||biospec||8/24/2015 1:26:27 AM||0.7||ng/ul||0.015||0.001||18.47||0.53||DNA||50.00
 +
|-
 +
|2||ptre||biospec||8/24/2015 1:29:38 AM||13.7||ng/ul||0.275||0.145||1.90||0.10||DNA||50.00
 +
|}
  
 +
<br>
  
 +
Digestion of “TEV-Protease pET45/pSB1C3 T7/pColA (24.08.2015)
 +
{| border="1" class="sortable"
 +
!Digestion
 +
|-
 +
|||DNA (500 ng/ul)||BamHI-HF||XhoI-HF||Cut Smart Buffer||ddH₂O||Total||
 +
|-
 +
|1x||5.86 ul||0.5 ul||0.5 ul||2.0 ul||11.14 ul||20.0 ul||
 +
|}
 +
<p>37˚C 3h</p>
  
 +
<br>
  
−
<h2 class="scroll">August</h2>
+
Ligation of “pCAGop with pTRE #12-2” (24.08.2015)
−
<div id="content-div">
+
{| border="1" class="wikitable"
 +
!Ligation
 +
|-
 +
|||pCAG (18.38 ng/ul)||pTRE #12-2 (100 ng/ul)||T4 DNA Ligase||T4 DNA Ligase Buffer||Cut Smart Buffer||ddH₂O||Total
 +
|-
 +
|1x||0.6 ul||6.6 ul||1.0 ul||2.0 ul||2.0 ul||9.8 ul||20.0 ul
 +
|}
 +
<p>Transformation was made.</p>
  
−
<p>11.08. We started designing our wiki.</p>
+
<br>
−
<p>18.08.2015 We got a little tired of doing experiments, so treated ourselves with an awesome team dinner at a fish restaurant.</p>
+
−
<p>22.08.2015 A team dinner again, but this time our dear professors Mehmet and Esra Gündüz hosted us at their house.</p>
+
−
<p>29.08.2015 Wiki’s tour part is drawn, now it’s time to color these drawings!</p>
+
−
</div>
+
  
−
  <a href="#" class="clickme" id="toggle" onclick="toggle_visibility('6');">Week 6<img src="https://static.igem.org/mediawiki/2014/f/f8/LMU14_arrow_down.png" id="6" class="tiger"></a>
+
Cut Check “pSB1C3-T7 #4,5,6,8” (24.08.2015)
 +
{| border="1" class="sortable"
 +
!Cut Check!!!!!!!!!!!!
 +
|-
 +
|||DNA (300 ng/ul)||EcoRI (FD)||PstI (FD)||Fast Digest Buffer||ddH₂O||Total
 +
|-
 +
|1x||3.5 ul||1.0 ul||1.0 ul||2.0 ul||12.5 ul||20.0 ul
 +
|}
 +
<p>30’ digestion</p>
  
 +
<br>
  
−
<div class="box">
 
−
 
−
Mini Prep of “pSB1C3-RFP”
 
−
</html>
 
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
 
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
 
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
 
|-
 
|-
−
|1||blank||biospec||8/1/2015 6:03:37 PM||-0.1||ng/ul||-0.003||-0.009||0.30||0.12||DNA||50.00
+
|1||blank||biospec||8/25/2015 1:26:12 PM||0.8||ng/ul||0.016||-0.003||-5.85||2.25||DNA||50.00
 
|-
 
|-
−
|2||psb1c3-A||biospec||8/1/2015 6:06:00 PM||207.0||ng/ul||4.140||2.184||1.90||2.11||DNA||50.00
+
|2||#9-5-4||biospec||8/25/2015 1:27:29 PM||59.1||ng/ul||1.182||0.603||1.96||2.25||DNA||50.00
 
|-
 
|-
−
|3||psb1c3-B||biospec||8/1/2015 6:07:07 PM||209.0||ng/ul||4.179||2.210||1.89||2.19||DNA||50.00
+
|3||#9-5-9||biospec||8/25/2015 1:28:30 PM||37.9||ng/ul||0.758||0.375||2.02||2.19||DNA||50.00
 +
|}
 +
 
 +
<br>
 +
 
 +
Colony PCR of “pTRE #13” (25.08.2015)
 +
{| border="1" class="wikitable"
 +
!Colony PCR
 
|-
 
|-
−
|4||psb1c3-C||biospec||8/1/2015 6:08:05 PM||157.3||ng/ul||3.147||1.664||1.89||2.09||DNA||50.00
+
|||MgCl₂||(NH₄)2SO₄||CMV fw||SV40 rv||dNTP||Tag||ddH₂O||DNA||Total
 
|-
 
|-
−
|5||psb1c3-D||biospec||8/1/2015 6:08:58 PM||242.5||ng/ul||4.851||2.548||1.90||2.11||DNA||50.00
+
|1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul
 
|-
 
|-
−
|6||psb1c3-E||biospec||8/1/2015 6:09:42 PM||116.4||ng/ul||2.329||1.215||1.92||2.17||DNA||50.00
+
|7x||17.5 ul||17.5 ul||7.0 ul||7.0 ul||3.5 ul||1.4 ul||86.1 ul||||140.0 ul
 +
|}
 +
{| border="1" class="wikitable"
 +
!Cycling
 
|-
 
|-
−
|7||psb1c3-F||biospec||8/1/2015 6:10:28 PM||236.4||ng/ul||4.729||2.503||1.89||2.17||DNA||50.00
+
|||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle
 
|-
 
|-
−
|8||psb1c3-71||biospec||8/1/2015 6:11:33 PM||111.7||ng/ul||2.233||1.175||1.90||2.10||DNA||50.00
+
|Time||5’||30’’||30’’||1.5’||5’||35x
 +
|}
 +
GEL GÖRÜNTÜSÜ + pTRE13 cut check görüntüsü
 +
 
 +
<br>
 +
 
 +
Colony PCR of “pCAG-DsRed” (25.08.2015)
 +
{| border="1" class="wikitable"
 +
!Colony PCR
 
|-
 
|-
−
|9||psb1c3-72||biospec||8/1/2015 6:12:14 PM||106.4||ng/ul||2.127||1.100||1.93||2.23||DNA||50.00
+
|||MgCl₂||(NH₄)2SO₄||pTRE Luc fw||SV40 rv||dNTP||Tag||ddH₂O||DNA||Total
 
|-
 
|-
−
|10||psb1c3-8||biospec||8/1/2015 6:12:51 PM||90.7||ng/ul||1.814||0.959||1.89||1.98||DNA||50.00
+
|1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul
 +
|-
 +
|14x||35.0 ul||35.0 ul||14.0 ul||14.0 ul||7.0 ul||2.8 ul||172.2 ul||||280.0 ul
 
|}
 
|}
 +
{| border="1" class="wikitable"
 +
!Cycling
 +
|-
 +
|||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle
 +
|-
 +
|Time||5’||30’’||30’’||1.5’||5’||35x
 +
|}
 +
<p>Result: Bands weren’t at the expected section.</p>
 +
GEL GÖRÜNTÜSÜ
  
−
<html>
 
 
<br>
 
<br>
  
−
Ligation of „pSB1C3 – Gblocks“ (02.08.2015)
+
Cut Check of „pSB1C3 – TlpB“ (25.05.2015)
−
</html>
+
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
−
!!!2!!3!!5!!6!!7!!8!!9!!10!!11!!12!!13!!14!!15!!
+
!Cut Check
 
|-
 
|-
−
|Insert||7.3 ul||7.0 ul||4.8 ul||8.4 ul||5.8 ul||8.3 ul||3.6 ul||3.9 ul||3.4 ul||4.4 ul||5.9 ul||3.9 ul||5.9 ul||
+
|||DNA (250 ng/ul)||EcoRI (FD)||Fast Digest Buffer||ddH₂O||Total||
 
|-
 
|-
−
|Vector||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||
+
|#8-1||3.6 ul||0.5 ul||2.0 ul||13.9 ul||20.0 ul||
 
|-
 
|-
−
|Buffer||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||
+
|#8-3||3.1 ul||0.5 ul||2.0 ul||14.4 ul||20.0 ul||
 +
|}
 +
<p>7˚C 35’</p>
 +
<p>The colonies will be made glycerol stock.</p>
 +
GEL GÖRÜNTÜSÜ
 +
 
 +
<br>
 +
 
 +
Ligation of “Damp-Pex” (25.08.2015)
 +
{| border="1" class="wikitable"
 +
!Ligation
 
|-
 
|-
−
|Enzyme||1.0 ul||1.0 ul||1.0 ul||1.0 ul||1.0 ul||1.0 ul||1.0 ul||1.0 ul||1.0 ul||1.0 ul||1.0 ul||1.0 ul||1.0 ul||
+
|||DNA (500 ng/ul)||NotI||Cut Smart Buffer||ddH₂O||Total||||
 
|-
 
|-
−
|ddH₂O||7.7 ul||8.0 ul||10.2 ul||6.6 ul||9.2 ul||6.7 ul||11.4 ul||11.1 ul||11.6 ul||10.6 ul||9.1 ul||11.1 ul||9.1 ul||
+
|#9-5||3.78 ul||1.0 ul||2.0 ul||13.22 ul||20.0 ul||||
 
|-
 
|-
−
|Total||20.0 ul||20.0 ul||20.0 ul||20.0 ul||20.0 ul||20.0 ul||20.0 ul||20.0 ul||20.0 ul||20.0 ul||20.0 ul||20.0 ul||20.0 ul||
+
|#9-10||3.87 ul||1.0 ul||2.0 ul||13.13 ul||20.0 ul||||
 
|}
 
|}
 +
<p>37˚C 3h</p>
  
 +
<br>
  
−
<html>
+
{| border="1" class="wikitable"
−
</div>
+
!Ligation
 +
|-
 +
|||ColA (100 ng/ul)||Insert (36 ng/ul)||T4 DNA Ligase||T4 DNA Ligase Buffer||ddH₂O||Total||
 +
|-
 +
|#9-5||10.0 ul||14.4 ul||1.0 ul||3.0 ul||1.6 ul||30.0 ul||
 +
|}
 +
<p>22˚C 1.5h</p>
 +
Transformation was made.
  
−
      <a href="#" class="clickme" id="toggle" onclick="toggle_visibility('7');">Week 7<img src="https://static.igem.org/mediawiki/2014/f/f8/LMU14_arrow_down.png" id="6" class="tiger"></a>
+
<br>
−
<div class="box">
+
  
−
Colony PCR of „pSB1C3 – Gblocks“ (03.08.2015)
+
Colony PCR of “pCAGop-DsRed” (26.08.2015)
−
</html>
+
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
 
!Colony PCR  
 
!Colony PCR  
 
|-
 
|-
−
|||MgCl₂||(NH₄)2SO₄||VR fwd||VR rv||dNTP||Tag||ddH₂O||DNA||Total
+
|||MgCl₂||(NH₄)2SO₄||pTRE Luc fw||SV40 rv||dNTP||Tag||ddH₂O||DNA||Total
 
|-
 
|-
 
|1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul
 
|1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul
 
|-
 
|-
−
|22X||55.0 ul||55.0 ul||22.0 ul||22.0 ul||11.0 ul||4.4 ul||271.0 ul||||440.4 ul
+
|10x||25.0 ul||25.0 ul||10.0 ul||10.0 ul||5.0 ul||2.0 ul||123.0 ul||||200.0 ul
 
|}
 
|}
−
 
 
{| border="1" class="wikitable"
 
{| border="1" class="wikitable"
 
!Cycling
 
!Cycling
Line 1,505: Line 1,822:
 
|Time||5’||30’’||30’’||1.5’||5’||35x
 
|Time||5’||30’’||30’’||1.5’||5’||35x
 
|}
 
|}
 +
<p>Result: 2 colonie will be made liquid culture and streak plate. Ligation and transformation will be repeated.</p>
 +
GEL GÖRÜNTÜSÜ
  
 +
<br>
  
−
<html>
+
Mini Prep of “pTRE #13-3,5” (26.08.2015)
 +
{| border="1" class="wikitable"
 +
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
 +
|-
 +
|1||blank||biospec||8/26/2015 4:55:28 PM||0.1||ng/ul||0.003||-0.020||-0.13||-0.13||DNA||50.00
 +
|-
 +
|2||ptre 13-3||biospec||8/26/2015 4:56:47 PM||77.9||ng/ul||1.558||0.772||2.02||1.67||DNA||50.00
 +
|-
 +
|3||ptre 13-5 ||biospec||8/26/2015 4:58:06 PM||76.0||ng/ul||1.520||0.776||1.96||1.48||DNA||50.00
 +
|}
 +
 
 +
<br>
 +
 
 +
Colony PCR of “pColA – Damp-Pex, pSB1C3 – TEV
 +
{| border="1" class="wikitable"
 +
!Colony PCR “pColA – Damp-Pex”
 +
|-
 +
|||MgCl₂||(NH₄)2SO₄||pTRE Luc fw||SV40 rv||dNTP||Tag||ddH₂O||DNA||Total
 +
|-
 +
|1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul
 +
|-
 +
|10x||25.0 ul||25.0 ul||10.0 ul||10.0 ul||5.0 ul||2.0 ul||123.0 ul||||200.0 ul
 +
|}
 +
{| border="1" class="wikitable"
 +
!Cycling
 +
|-
 +
|||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle
 +
|-
 +
|Time||5’||30’’||30’’||1.5’||5’||35x
 +
|}
 +
 +
<br>
 +
 +
Colony PCR “pSB1C3 – TEV Protease”
 +
{| border="1" class="wikitable"
 +
!Colony PCR “pSB1C3 – TEV Protease”
 +
|-
 +
|||MgCl₂||(NH₄)2SO₄||VR||VF||dNTP||Tag||ddH₂O||DNA||Total
 +
|-
 +
|1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul
 +
|-
 +
|10x||25.0 ul||25.0 ul||10.0 ul||10.0 ul||5.0 ul||2.0 ul||123.0 ul||||200.0 ul
 +
|}
 +
{| border="1" class="wikitable"
 +
!Cycling
 +
|-
 +
|||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle
 +
|-
 +
|Time||5’||30’’||30’’||1.5’||5’||35x
 +
|}
 +
<p>Result: negative</p>
 +
GEL GÖRÜNTÜSÜ
 +
 +
<br>
 +
 +
Colony PCR “pSB1C3 – TEV Protease” – Repeat (27.08.2015)
 +
{| border="1" class="wikitable"
 +
!Colony PCR “pSB1C3 – TEV Protease”
 +
|-
 +
|||MgCl₂||(NH₄)2SO₄||VR||VF||dNTP||Tag||ddH₂O||DNA||Total
 +
|-
 +
|1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul
 +
|-
 +
|15x||37.5 ul||37.5 ul||15.0 ul||15.0 ul||7.5 ul||3.0 ul||184.5 ul||||300.0 ul
 +
|}
 +
{| border="1" class="wikitable"
 +
!Cycling
 +
|-
 +
|||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle
 +
|-
 +
|Time||5’||30’’||30’’||1.5’||5’||35x
 +
|}
 +
<p>Result: negative</p>
 +
GEL GÖRÜNTÜSÜ
 +
 +
<br>
 +
 +
Colony PCR of “pTRE – Luc #14” (27.08.2015)
 +
{| border="1" class="wikitable"
 +
!Colony PCR
 +
|-
 +
|||MgCl₂||(NH₄)2SO₄||pTRE Luc fw||pTRE Luc rv||dNTP||Tag||ddH₂O||DNA||Total
 +
|-
 +
|1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul
 +
|-
 +
|14x||35.0 ul||35.0 ul||14.0 ul||14.0 ul||7.0 ul||2.8 ul||172.2 ul||||280.0 ul
 +
|}
 +
{| border="1" class="wikitable"
 +
!Cycling
 +
|-
 +
|||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle
 +
|-
 +
|Time||5’||30’’||30’’||1.5’||5’||35x
 +
|}
 +
<p>Result: Bands weren’t at the expected section.</p>
 +
GEL GÖRÜNTÜSÜ
 +
 +
<br>
 +
 +
Gel Extraction of “Damp-Pex”
 +
{| border="1" class="wikitable"
 +
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
 +
|-
 +
|1||blank||biospec||8/27/2015 9:39:17 PM||1.1||ng/ul||0.022||0.009||2.36||1.00||DNA||50.00
 +
|-
 +
|2||damp-pex||biospec||8/27/2015 9:40:04 PM||8.2||ng/ul||0.164||0.090||1.82||0.05||DNA||50.00
 +
|}
 +
 +
<br>
 +
 +
Colony PCR of “pCAGop-DsRed #12” (27.08.2015)
 +
{| border="1" class="wikitable"
 +
!Colony PCR
 +
|-
 +
|||MgCl₂||(NH₄)2SO₄||pTRE Luc fw||SV40 rv||dNTP||Tag||ddH₂O||DNA||Total
 +
|-
 +
|1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul
 +
|-
 +
|14x||35.0 ul||35.0 ul||14.0 ul||14.0 ul||7.0 ul||2.8 ul||172.2 ul||||280.0 ul
 +
|}
 +
{| border="1" class="wikitable"
 +
!Cycling
 +
|-
 +
|||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle
 +
|-
 +
|Time||5’||30’’||30’’||1.5’||5’||35x
 +
|}
 +
<p>Result: Negative.</p>
 +
GEL GÖRÜNTÜSÜ
 +
 +
<br>
 +
 +
Ligation of “pCAGop-DsRed” (27.08.2015)
 +
{| border="1" class="wikitable"
 +
!Ligation
 +
|-
 +
|||pCAG (18.38 ng/ul)||pTRE #12 (100 ng/ul)||T4 DNA Ligase||T4 DNA Ligase Buffer||ddH₂O||Total
 +
|-
 +
|1x||0.3 ul||6.6 ul||1.0 ul||2.0 ul||10.1 ul||20.0 ul
 +
|}
 +
<p>22˚C 1.30h</p>
 +
<p>Transformation was made.</p>
 +
 +
<br>
 +
 +
Colony PCR of „pSB1C3-T7 #5“ (27.08.2015)
 +
{| border="1" class="wikitable"
 +
!Colony PCR
 +
|-
 +
|||MgCl₂||(NH₄)2SO₄||pTRE Luc fw||SV40 rv||dNTP||Tag||ddH₂O||DNA||Total
 +
|-
 +
|1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul
 +
|-
 +
|8x||20.0 ul||20.0 ul||8.0 ul||8.0 ul||4.0 ul||1.6 ul||98.4 ul||||160.0 ul
 +
|}
 +
{| border="1" class="wikitable"
 +
!Cycling
 +
|-
 +
|||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle
 +
|-
 +
|Time||5’||30’’||30’’||1.5’||5’||35x
 +
|}
 +
<p>Result: #1-1 Liquid Culture was made.</p>
 +
GEL GÖRÜNTÜSÜ
 +
 +
<br>
 +
 +
Digestion of “pSB1C3-T7 TEV Protease (17. and 18. Colony)” (27.08.2015)
 +
{| border="1" class="sortable"
 +
!Digestion
 +
|-
 +
|||DNA (500 ng/ul)||NotI-HF||Cut Smart Buffer||ddH₂O||Total
 +
|-
 +
|17||6.0 ul||0.5 ul||2.0 ul||11.5 ul||20.0 ul
 +
|-
 +
|18||13.0 ul||0.5 ul||2.0 ul||4.5 ul||20.0 ul
 +
|}
 +
<p>37˚C 3h</p>
 +
<p>65˚C 20’’ Heat Inactivation</p>
 +
<p>Result: negative</p>
 +
GEL GÖRÜNTÜSÜ
 +
 
 +
<br>
 +
 +
Digestion of “pColA” (27.08.2015)
 +
{| border="1" class="wikitable"
 +
!Digestion
 +
|-
 +
|||pColA (300 ng/ul)||NotI||Buffer||ddH₂O||Total
 +
|-
 +
|1x||11.76 ul||0.5 ul||2.0 ul||5.74 ul||20.0 ul
 +
|}
 +
<p>37˚C 3h</p>
 +
<p>1.2 ul rSAP</p>
 +
<p>37˚C 1h</p>
 +
<p>20’’ Heat Inactivation</p>
 +
 +
<br>
 +
 +
Colony PCR of “pCAGop-DsRed #12-2. Colony streak” (28.08.2015)
 +
{| border="1" class="wikitable"
 +
!Colony PCR
 +
|-
 +
|||MgCl₂||(NH₄)2SO₄||pTRE Luc fw||SV40 rv||dNTP||Tag||ddH₂O||DNA||Total
 +
|-
 +
|1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul
 +
|-
 +
|12x||30.0 ul||30.0 ul||12.0 ul||12.0 ul||6.0 ul||2.4 ul||147.6 ul||||240.0 ul
 +
|}
 +
{| border="1" class="wikitable"
 +
!Cycling
 +
|-
 +
|||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle
 +
|-
 +
|Time||5’||30’’||30’’||1.5’||5’||35x
 +
|}
 +
<p>Result: Negative</p>
 +
GEL GÖRÜNTÜSÜ
 +
 +
<br>
 +
 +
Colony PCR of „pSB1C3-T7 #5“ (28.08.2015)
 +
{| border="1" class="wikitable"
 +
!Colony PCR
 +
|-
 +
|||MgCl₂||(NH₄)2SO₄||VF||VR||dNTP||Tag||ddH₂O||DNA||Total
 +
|-
 +
|1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul
 +
|-
 +
|16x||40.0 ul||40.0 ul||16.0 ul||16.0 ul||13.0 ul||3.2 ul||196.8 ul||||325.0 ul
 +
|}
 +
{| border="1" class="wikitable"
 +
!Cycling
 +
|-
 +
|||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle
 +
|-
 +
|Time||5’||30’’||30’’||1.5’||5’||35x
 +
|}
 +
<p>Result: #5-31 Bands were at the expected section</p>
 +
GEL GÖRÜNTÜSÜ
 +
 +
<br>
 +
 +
Cut Check of „pSB1C3-T7 #5-31“
 +
{| border="1" class="wikitable"
 +
!Cut Check
 +
|-
 +
|||DNA||EcoRI (FD)||PstI (FD)||Fast Digest Buffer||ddH₂O||Total
 +
|-
 +
|1x||???????||1.0 ul||1.0 ul||2.0 ul||?????||20.0 ul
 +
|}
 +
GEL GÖRÜNTÜSÜ
 +
 +
Digestion of “pSB1C3 #9” (28.08.2015)
 +
{| border="1" class="wikitable"
 +
!Digestion
 +
|-
 +
|||DNA (500 ng/ul)||NotI||Cut Smart Buffer||ddH₂O||Total||
 +
|-
 +
|1x||???????||1.0 ul||2.0 ul||?????||20.0 ul||
 +
|}
 +
<p>3h was digested</p>
 +
<p>Result: #9-5 Bands were at the expected section</p>
 +
GEL GÖRÜNTÜSÜ
 +
 +
<br>
 +
 +
Ligation of “pColA #9”
 +
{| border="1" class="wikitable"
 +
!Ligation
 +
|-
 +
|||pColA||#9||T4 DNA Ligase||T4 DNA Ligase Buffer||ddH₂O||Total
 +
|-
 +
|1x||7.5 ul||9.2 ul||1.0 ul||2.0 ul||0.3 ul||20.0 ul
 +
|}
 +
<p>2h was ligasted</p>
 +
 +
<br>
 +
 +
Mini Prep of “pCAG-DsRed #12” (28.08.2015)
 +
{| border="1" class="wikitable"
 +
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
 +
|-
 +
|1||blank||biospec||8/28/2015 7:23:31 PM||0.5||ng/ul||0.010||-0.006||-1.58||-1.46||DNA||50.00
 +
|-
 +
|2||pcag op-dsred||biospec||8/28/2015 7:24:38 PM||92.3||ng/ul||1.846||1.105||1.67||0.79||DNA||50.00
 +
|-
 +
|3||pcag op-dsred||biospec||8/28/2015 7:26:20 PM||67.4||ng/ul||1.348||0.702||1.92||1.35||DNA||50.00
 +
|}
 +
 +
<br>
 +
 +
Cut Check “pCAGop-DsRed #12”
 +
{| border="1" class="wikitable"
 +
!Cut Check
 +
|-
 +
|||DNA||EcoRI-HF||XhoI (NEB)||Cut Smart Buffer||ddH₂O||Total
 +
|-
 +
|1x||3.7 ul||0.5 ul||0.5 ul||2.0 ul||13.3 ul||20.0 ul
 +
|}
 +
 +
<p>Result: negative</p>
 +
 +
GEL GÖRÜNTÜSÜ
 +
 +
<br>
 +
 +
Cut Check “pCAGop-DsRed #12” - Repeat
 +
{| border="1" class="wikitable"
 +
!Cut Check
 +
|-
 +
|||DNA||EcoRI-HF||XhoI (NEB)||Cut Smart Buffer||ddH₂O||Total
 +
|-
 +
|1x||3.7 ul||0.5 ul||0.5 ul||2.0 ul||13.3 ul||20.0 ul
 +
|}
 +
<p>Result: negative</p>
 +
 +
<br>
 +
 +
Colony PCR of „pTEToff and pTEToff N term“ (28.08.2015)
 +
{| border="1" class="wikitable"
 +
!Colony PCR
 +
|-
 +
|||MgCl₂||(NH₄)2SO₄||CMV rv||pTRE Luc fw||dNTP||Tag||ddH₂O||DNA||Total
 +
|-
 +
|1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul
 +
|-
 +
|26x||65.0 ul||65.0 ul||26.0 ul||26.0 ul||13.0 ul||5.2 ul||319.8 ul||||520.0 ul
 +
|}
 +
{| border="1" class="wikitable"
 +
!Cycling
 +
|-
 +
|||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle
 +
|-
 +
|Time||5’||30’’||30’’||1.5’||5’||35x
 +
|}
 +
<p>Result: positive</p>
 +
GEL GÖRÜNTÜSÜ
 +
 +
<br>
 +
 +
Mini Prep of “pSB1C3-T7 TEV Protease” (28.09.2015)
 +
{| border="1" class="wikitable"
 +
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
 +
|-
 +
|1||blank||biospec||8/28/2015 5:55:09 PM||0.0||ng/ul||-0.001||-0.010||0.10||-1.29||DNA||50.00
 +
|-
 +
|2||tev  psb1c3-t7/17||biospec||8/28/2015 5:56:45 PM||81.8||ng/ul||1.637||0.848||1.93||2.08||DNA||50.00
 +
|-
 +
|3||tev  psb1c3-t7/18||biospec||8/28/2015 5:59:01 PM||38.5||ng/ul||0.771||0.380||2.03||2.03||DNA||50.00
 +
|}
 +
 +
<br>
 +
 +
Mini Prep of “pSB1C3-T7 TEV Protease” (29.09.2015)
 +
{| border="1" class="wikitable"
 +
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
 +
|-
 +
|1||blank||biospec||8/29/2015 8:08:11 PM||-0.8||ng/ul||-0.016||-0.019||0.83||0.68||DNA||50.00
 +
|-
 +
|2||psb1c3-t7/tev17||biospec||8/29/2015 8:09:26 PM||135.4||ng/ul||2.709||1.355||2.00||1.68||DNA||50.00
 +
|-
 +
|3||psb1c3-t7/tev18||biospec||8/29/2015 8:11:06 PM||229.1||ng/ul||4.581||2.193||2.09||2.40||DNA||50.00
 +
|}
 +
 +
<br>
 +
 +
Mini Prep of “pCAGop-DsRed #1-1” (30.08.2015)
 +
{| border="1" class="wikitable"
 +
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
 +
|-
 +
|1||blank||biospec||8/30/2015 11:05:33 AM||0.2||ng/ul||0.003||-0.012||-0.25||-0.27||DNA||50.00
 +
|-
 +
|2||pcag op-dsred||biospec||8/30/2015 11:07:31 AM||79.8||ng/ul||1.595||0.823||1.94||1.72||DNA||50.00
 +
|}
 +
 +
<br>
 +
 +
Cut Check of “pCAGop-DsRed #1-1”
 +
{| border="1" class="wikitable"
 +
!Cut Check
 +
|-
 +
|||pCAGop-DsRed (250 ng/ul)||EcoRI-HF||XhoI (NEB)||Cut Smart Buffer||ddH₂O||Total
 +
|-
 +
|1x||3.2 ul||0.5 ul||0.5 ul||2.0 ul||13.8 ul||20.0 ul
 +
|}
 +
<p>37˚C 3h</p>
 +
<p>Result: negative</p>
 +
GEL GÖRÜNTÜSÜ
 +
 +
<br>
 +
 +
Digestion of “pSB1C3-T7 TEV Protease” (30.08.2015)
 +
{| border="1" class="wikitable"
 +
!Digestion
 +
|-
 +
|||Insert||NotI||Cut Smart Buffer||ddH₂O||Total
 +
|-
 +
|17||3.7 ul||0.25 ul||2.0 ul||14.05 ul||20.0 ul
 +
|-
 +
|18||2.2 ul||0.25 ul||2.0 ul||15.55 ul||20.0 ul
 +
|}
 +
<p>37˚C 3h</p>
 +
<p>Result: Bands were at the expected section. Gel extraction was made.</p>
 +
GEL GÖRÜNTÜSÜ
 +
 +
<br>
 +
 +
Ligation of “pSB1C3-T7 TEV Protease”
 +
{| border="1" class="wikitable"
 +
!Ligation
 +
|-
 +
|||Vector (60 ng/ul)||Insert (29.11 ng/ul)||T4 DNA Buffer||T4 DNA Buffer Ligase||ddH₂O||Total
 +
|-
 +
|1x||7.0 ul||8.7 ul||2.0 ul||1.0 ul||1.3 ul||20.0 ul
 +
|}
 +
<p>22˚C 1.5h</p>
 +
<p>Transformation was made.</p>
 +
 +
<br>
 +
 +
Gel Extraction
 +
{| border="1" class="wikitable"
 +
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
 +
|-
 +
|1||blank||biospec||8/30/2015 5:28:51 PM||0.0||ng/ul||-0.001||-0.005||0.15||0.03||DNA||50.00
 +
|-
 +
|2||psb1c3-t7||biospec||8/30/2015 5:28:05 PM||5.6||ng/ul||0.113||0.061||1.85||0.17||DNA||50.00
 +
|}
 +
 +
<br>
 +
 +
Colony PCR of “pColA – TEV” (31.08.2015)
 +
{| border="1" class="wikitable"
 +
!Colony PCR
 +
|-
 +
|||MgCl₂||(NH₄)2SO₄||pColA fw||pColA rv||dNTP||Tag||ddH₂O||DNA||Total
 +
|-
 +
|1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul
 +
|-
 +
|16x||40.0 ul||40.0 ul||16.0 ul||16.0 ul||8.0 ul||3.2 ul||196.8 ul||||320.0 ul
 +
|}
 +
{| border="1" class="wikitable"
 +
!Cycling
 +
|-
 +
|||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle
 +
|-
 +
|Time||5’||30’’||30’’||1.5’||5’||35x
 +
|}
 +
<p>Result: negative</p>
 +
GEL GÖRÜNTÜSÜ
 +
 +
<br>
 +
 +
Colony PCR of “pColA – TEV” – Repeat (31.08.2015)
 +
{| border="1" class="wikitable"
 +
!Colony PCR
 +
|-
 +
|||MgCl₂||(NH₄)2SO₄||pColA fw||pColA rv||dNTP||Tag||ddH₂O||DNA||Total
 +
|-
 +
|1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul
 +
|-
 +
|22x||55.0 ul||55.0 ul||22.0 ul||22.0 ul||11.0 ul||3.2 ul||270.6 ul||||440.0 ul
 +
|}
 +
{| border="1" class="wikitable"
 +
!Cycling
 +
|-
 +
|||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle
 +
|-
 +
|Time||5’||30’’||30’’||1.5’||5’||35x
 +
|}
 +
<p>Result: negative</p>
 +
GEL GÖRÜNTÜSÜ
 +
 +
<br>
 +
 +
Digestion of “PsicA for pSB1C3 and invF-sicA for ColA” (31.08.2015)
 +
{| border="1" class=wikitable"
 +
!Digestion of “PsicA and invF”
 +
|-
 +
|||DNA||EcoRI-HF||PstI||XbaI||SpeI||2.1 Buffer||Cut Smart Buffer||ddH₂O||Total
 +
|-
 +
|invF-sicA||10.0 ul||-||-||1.0 ul||1.0 ul||-||2.0 ul||6.0 ul||20.0 ul
 +
|-
 +
|PsicA||10.0 ul||1.0 ul||1.0 ul||-||-||2.0 ul||-||6.0 ul||20.0 ul
 +
|}
 +
<p>37˚C 10h</p>
 +
<p>82˚C 20’ Heat Inactivation</p>
 +
 +
<br>
 +
 +
{| border="1" class="wikitable"
 +
!Digestion of “pSB1C3-RFP and pColA-NotI”
 +
|-
 +
|||DNA||EcoRI (FD)||PstI (FD)||XbaI (FD)||SpeI (FD)||Fast Digest Buffer||ddH₂O||Total||
 +
|-
 +
|pSB1C3-RFP (1000 ng/ul)||2.5 ul||1.0 ul||1.0 ul||-||-||2.0 ul||13.5 ul||20.0 ul||
 +
|-
 +
|pColA-NotI (500 ng/ul)||5.0 ul||-||-||1.0 ul||1.0 ul||2.0 ul||11.0 ul||20.0 ul||
 +
|}
 +
 +
<html>
 +
 +
</section>
 +
 +
<html>
 +
 
</div>
 
</div>
 +
 +
</section>
 +
 
</div>
 
</div>
 +
 +
</div>
 +
 +
<h2 class="scroll">September</h2>
 +
<div id="content-div">
 +
 +
</html>
 +
 +
02.09.2015 Wiki meetings continue, we are making decisions together.
 +
06.09.2015 What will we be wearing at the Giant Jamboree? Thinking about how to design our T-shirts.
 +
08.09.2015 Team pictures mustn’t be left ends.
 +
11.09.2015 We taught our little friends synthetic biology. Those kids were really funny when they came first, they were covering their faces because they were scared of being contaminated.
 +
13.09.2015 Coloring our drawings is complete. They are better than original now. We still have wiki meetings, there are only 5 days to wiki-freeze!
 +
 +
<html>
 +
 +
    <div style="margin-bottom:60px">
 +
    <section class="accordion">
 +
          <div>       
 +
            <input id="september-week1" name="september-week1" type="checkbox" />
 +
            <label for="september-week1">Week 1, 01.09.-06.09.</label>
 +
            <article class="ac-small">
 +
 +
</html>
 +
 +
Ligation of “ColA and pSB1C3” (01.09.2015)
 +
{| border="1" class="wikitable"
 +
!Ligation
 +
|-
 +
|||Vector (100 ng/ul)||invF-sicA||psicA||T4 DNA Ligase||T4 DNA Ligase Buffer||ddH₂O||Total
 +
|-
 +
|ColA||8.5 ul||17.5 ul||-||1.0 ul||2.0 ul||-||30.0 ul
 +
|-
 +
|pSB1C3||9.44 ul||-||13.55 ul||1.0 ul||2.0 ul||3.0 ul||30.0 ul
 +
|}
 +
<p>22˚C 2h</p>
 +
 +
<br>
 +
{| border="1" class="wikitable"
 +
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
 +
|-
 +
|1||blank||biospec||9/1/2015 4:10:17 AM||0.3||ng/ul||0.006||-0.007||-0.88||-0.78||DNA||50.00
 +
|-
 +
|2||colA ext||biospec||9/1/2015 4:11:28 AM||11.7||ng/ul||0.235||0.132||1.78||0.08||DNA||50.00
 +
|-
 +
|3||cola clean-up||biospec||9/1/2015 4:12:56 AM||7.2||ng/ul||0.143||0.078||1.84||0.06||DNA||50.00
 +
|-
 +
|4||psb1c3 ext||biospec||9/1/2015 4:14:40 AM||10.6||ng/ul||0.213||0.113||1.88||0.12||DNA||50.00
 +
|}
 +
<p>Transformation will be made.</p>
 +
 +
<br>
 +
 +
Ligation of “pCAGop-DsRed” (01.09.2015)
 +
{| border="1" class=wikitable"
 +
!Ligation
 +
|-
 +
|||pCAG||pTRE #12||T4 DNA Ligase||T4 DNA Ligase Buffer||ddH₂O||Total||
 +
|-
 +
|1x||0.3 ul||4.0 ul||1.0 ul||1.0 ul||13.7 ul||20.0 ul||
 +
|}
 +
<p>Transformation with BL21.</p>
 +
 +
<br>
 +
 +
Digestion of “pSB1C3-T7 TEV and pColA” (01.09.2015)
 +
{| border="1" class="wikitable"
 +
!Digestion
 +
|-
 +
|||DNA||XhoI-HF||SpeI-HF||Cut Smart Buffer||RNAse||ddH₂O||Total
 +
|-
 +
|17 (Hand Made Solution)||10.0 ul||0.25 ul||0.25 ul||2.0 ul||0.2 ul||7.3 ul||20.0 ul
 +
|-
 +
|18 (Hand Made Solution)||10.0 ul||0.25 ul||0.25 ul||2.0 ul||0.2 ul||7.3 ul||20.0 ul
 +
|-
 +
|17 (Midi Prep Solution)||10.0 ul||0.25 ul||0.25 ul||2.0 ul||-||7.5 ul||20.0 ul
 +
|-
 +
|18 (Midi Prep Solution)||10.0 ul||0.25 ul||0.25 ul||2.0 ul||-||7.5 ul||20.0 ul
 +
|-
 +
|pColA (1000 ng/ul)||9.0 ul||0.5 ul||0.5 ul||2.0 ul||-||8.0 ul||
 +
|}
 +
<p>37˚C 3h</p>
 +
<p>80˚C 20’ Heat Inactivation</p>
 +
<p>Cut Check was made.</p>
 +
Görüntü
 +
<p>Result: pColA Bands were at the expected section. Midi Prep Solution is better.</p>
 +
 +
<br>
 +
 +
Colony PCR of “pCAGop-DsRed and pCMV-lacO” (02.09.2015)
 +
{| border="1" class="wikitable"
 +
!Colony PCR
 +
|-
 +
|||MgCl₂||(NH₄)2SO₄||pTRE Luc fw||SV40 rv||dNTP||Tag||ddH₂O||DNA||Total
 +
|-
 +
|1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul
 +
|-
 +
|16x||40.0 ul||40.0 ul||16.0 ul||16.0 ul||8.0 ul||3.2 ul||196.8 ul||||320.0 ul
 +
|}
 +
{| border="1" class="wikitable"
 +
!Cycling
 +
|-
 +
|||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle
 +
|-
 +
|Time||5’||30’’||30’’||1.5’||5’||35x
 +
|}
 +
<p>Result: negative. It will be repeated.</p>
 +
GEL GÖRÜNTÜSÜ
 +
 +
<br>
 +
 +
Colony PCR of “invF-sicA and PsicA” (02.09.2015)
 +
<p>Result: PsicA: 1, 2, 6, 8, 9, 10, 11 Liquid Culture will be made.</p>
 +
GEL GÖRÜNTÜSÜ
 +
 +
<br>
 +
 +
Digestion of “invF-sicA for pSB1C3 and ColA” (02.09.2015)
 +
<p>DNA 10.0 ul + rSAP 1.0 ul / 37˚C 1h / 80˚C 20’</p>
 +
{| border="1" class="wikitable"
 +
!Digestion
 +
|-
 +
|||DNA||EcoRI-HF||PstI||XbaI||SpeI||2.1 Buffer||Cut Smart Buffer||ddH₂O||Total
 +
|-
 +
|pSB1C3||10.0 ul||0.4 ul||0.4 ul||-||-||2.0 ul||-||7.2 ul||20.0 ul
 +
|-
 +
|ColA||10.0 ul||-||-||0.4 ul||0.4 ul||-||2.0 ul||7.2 ul||20.0 ul
 +
|}
 +
<p>37˚C overnight</p>
 +
 +
<br>
 +
 +
Mini Prep of “PsicA – pSB1C3” (03.08.2015)
 +
{| border="1" class="wikitable"
 +
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
 +
|-
 +
|1||blank||biospec||9/3/2015 8:34:29 PM||0.8||ng/ul||0.016||0.000||116.17||0.85||DNA||50.00
 +
|-
 +
|2||psb1c3-psica 1||biospec||9/3/2015 8:35:32 PM||130.5||ng/ul||2.610||1.370||1.91||2.04||DNA||50.00
 +
|-
 +
|3||psb1c3-psica 2||biospec||9/3/2015 8:36:34 PM||140.9||ng/ul||2.818||1.457||1.93||2.16||DNA||50.00
 +
|-
 +
|4||psb1c3-psica 6||biospec||9/3/2015 8:37:28 PM||178.5||ng/ul||3.569||1.864||1.92||2.20||DNA||50.00
 +
|-
 +
|5||psb1c3-psica 8||biospec||9/3/2015 8:38:24 PM||98.7||ng/ul||1.974||1.021||1.93||2.13||DNA||50.00
 +
|-
 +
|6||psb1c3-psica 9||biospec||9/3/2015 8:39:21 PM||237.0||ng/ul||4.741||2.476||1.91||2.13||DNA||50.00
 +
|-
 +
|7||psb1c3-psica 10||biospec||9/3/2015 8:40:35 PM||109.0||ng/ul||2.181||1.127||1.94||2.05||DNA||50.00
 +
|-
 +
|8||||biospec||9/3/2015 8:41:51 PM||109.5||ng/ul||2.189||1.134||1.93||1.97||DNA||50.00
 +
|-
 +
|9||blank||biospec||9/3/2015 8:43:10 PM||1.3||ng/ul||0.025||0.003||9.26||0.73||DNA||50.00
 +
|-
 +
|10||psb1c3-psica 11||biospec||9/3/2015 8:44:14 PM||50.4||ng/ul||1.008||0.518||1.95||1.97||DNA||50.00
 +
|}
 +
 +
<br>
 +
 +
Cut Check of “PsicA – pSB1C3”
 +
{| border="1" class="wikitable"
 +
!Cut Check
 +
|-
 +
|||DNA||EcoRI (FD)||PstI (FD)||Fast Digest Buffer||ddH₂O||Total
 +
|-
 +
|1||1.9 ul||0.35 ul||0.35 ul||2.0 ul||15.4 ul||20.0 ul
 +
|-
 +
|2||1.8 ul||0.35 ul||0.35 ul||2.0 ul||15.5 ul||20.0 ul
 +
|-
 +
|6||1.4 ul||0.35 ul||0.35 ul||2.0 ul||15.9 ul||20.0 ul
 +
|-
 +
|8||2.5 ul||0.35 ul||0.35 ul||2.0 ul||14.8 ul||20.0 ul
 +
|-
 +
|9||1.1 ul||0.35 ul||0.35 ul||2.0 ul||16.2 ul||20.0 ul
 +
|-
 +
|10||2.3 ul||0.35 ul||0.35 ul||2.0 ul||15.0 ul||20.0 ul
 +
|-
 +
|11||5.0 ul||0.35 ul||0.35 ul||2.0 ul||12.3 ul||20.0 ul
 +
|}
 +
<p>37˚C 35’</p>
 +
 +
<br>
 +
 +
Ligation of “invF and pSB1C3/pColA” (03.08.2015)
 +
{| border="1" class="wikitable"
 +
!Ligation!!!!!!!!!!!!!!
 +
|-
 +
|||Vector (100 ng/ul)||invF/sicA||invF/sicA||T4 DNA Ligase||T4 DNA Ligase Buffer||ddH₂O||Total
 +
|-
 +
|ColA (-)||11.6 ul||-||-||1.0 ul||3.2 ul||16.6 ul||32.2 ul
 +
|-
 +
|ColA (+)||11.6 ul||14.40 ul||-||3.2 ul||3.4 ul||2.2 ul||34.8 ul
 +
|-
 +
|pSB1C3||9.4 ul||-||14.40 ul||1.0 ul||3.0 ul||2.2 ul||30.0 ul
 +
|}
 +
<p>RT 1h </p>
 +
<p>Transformation will be made.</p>
 +
 +
<br>
 +
 +
Colony PCR of “pCMV-Lacop-DsRed and pCAGop-DsRed” (04.09.2015)
 +
{| border="1" class="wikitable"
 +
!Colony PCR
 +
|-
 +
|||MgCl₂||(NH₄)2SO₄||pTRE Luc fw||SV40 rv||dNTP||Tag||ddH₂O||DNA||Total
 +
|-
 +
|1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul
 +
|-
 +
|15x||37.5 ul||37.5 ul||15.0 ul||15.0 ul||7.5 ul||3.0 ul||184.5 ul||||300.0 ul
 +
|}
 +
{| border="1" class="wikitable"
 +
!Cycling
 +
|-
 +
|||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle
 +
|-
 +
|Time||5’||30’’||30’’||1.5’||5’||35x
 +
|}
 +
<p>Result: Bands were at the expected section. Liquid Culture was made.</p>
 +
GEL GÖRÜNTÜSÜ
 +
 +
<br>
 +
 +
Mini Prep of „pCMV-Lacop-DsRed and pCAGop-DsRed“ (05.09.2015)
 +
{| border="1" class="wikitable"
 +
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
 +
|-
 +
|1||blank||biospec||9/5/2015 10:42:13 AM||-0.1||ng/ul||-0.003||-0.015||0.18||0.15||DNA||50.00
 +
|-
 +
|2||pcmv-lacop-dsred 1||biospec||9/5/2015 10:44:11 AM||68.4||ng/ul||1.369||0.690||1.98||1.76||DNA||50.00
 +
|-
 +
|3||pcmv-lacop-dsred 2||biospec||9/5/2015 10:45:12 AM||51.2||ng/ul||1.024||0.499||2.05||1.77||DNA||50.00
 +
|-
 +
|4||pcmv-lacop-dsred 3||biospec||9/5/2015 10:46:09 AM||78.5||ng/ul||1.570||0.793||1.98||1.98||DNA||50.00
 +
|-
 +
|5||pcmv-lacop-dsred 4||biospec||9/5/2015 10:47:04 AM||47.7||ng/ul||0.954||0.460||2.07||1.95||DNA||50.00
 +
|-
 +
|6||Pcagop-dsred 1||biospec||9/5/2015 10:48:08 AM||499.6||ng/ul||9.991||5.290||1.89||2.20||DNA||50.00
 +
|}
 +
 +
<br>
 +
 +
Cut Check of „pCMV-Lacop-DsRed and pCAGop-DsRed“
 +
{| border="1" class="wikitable"
 +
!Cut Check
 +
|-
 +
|||DNA||BamHI||XhoI||EcoRI||Cut Smart Buffer||ddH₂O||Total
 +
|-
 +
|1||4.4 ul||0.5 ul||0.5 ul||-||2.0 ul||12.6 ul||20.0 ul
 +
|-
 +
|2||5.9 ul||0.5 ul||0.5 ul||-||2.0 ul||11.1 ul||20.0 ul
 +
|-
 +
|3||3.9 ul||0.5 ul||0.5 ul||-||2.0 ul||13.1 ul||20.0 ul
 +
|-
 +
|4||6.3 ul||0.5 ul||0.5 ul||-||2.0 ul||10.7 ul||20.0 ul
 +
|-
 +
|pCAGop-DsRed||1.0 ul||0.5 ul||-||0.5 ul||2.0 ul
 +
|-
 +
|||16.0 ul||20.0 ul
 +
|}
 +
<p>37˚C 3h</p>
 +
<p>Result: 1-2-3-4 is positive.</p>
 +
GEL GÖRÜNTÜSÜ
 +
 +
 +
 +
</div>
 +
<html>
 +
</section>
 +
 +
      <section class="accordion">
 +
          <div>
 +
            <input id"september-week2" name="september-week2" type="checkbox" />
 +
            <label for="september-week2">Week 2, 07.09.-13.09.</label>
 +
            <article class="ac-small">
 +
</html>
 +
 +
Cut Check of “invF and sicA” (07.09.2015)
 +
{| border="1" class="wikitable"
 +
!Cut Check
 +
|-
 +
|||DNA (250 ng/ul)||EcoRI (FD)||PstI (FD)||Fast Digest Buffer||ddH₂O||Total||
 +
|-
 +
|1x||5.0 ul||0.5 ul||0.5 ul||2.0 ul||12.0 ul||20.0 ul||
 +
|}
 +
<p>37˚C 35’</p>
 +
GEL GÖRÜNTÜSÜ
 +
 +
<br>
 +
 +
Mini Prep
 +
{| border="1" class="wikitable"
 +
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
 +
|-
 +
|1||blank||biospec||9/7/2015 6:34:56 PM||-0.1||ng/ul||-0.003||-0.005||0.60||0.11||DNA||50.00
 +
|-
 +
|2||#18-11||biospec||9/7/2015 6:36:20 PM||79.8||ng/ul||1.597||0.843||1.89||1.93||DNA||50.00
 +
|-
 +
|3||#18-2||biospec||9/7/2015 6:37:48 PM||74.9||ng/ul||1.497||0.787||1.90||2.01||DNA||50.00
 +
|-
 +
|4||elution blank ||biospec||9/7/2015 6:39:44 PM||0.3||ng/ul||0.005||-0.004||-1.51||0.68||DNA||50.00
 +
|-
 +
|5||#18-5 elution||biospec||9/7/2015 6:40:49 PM||57.7||ng/ul||1.154||0.604||1.91||1.91||DNA||50.00
 +
|}
 +
 +
<br>
 +
 +
Digestion of “#18-2,5”
 +
{| border="1" class="wikitable"
 +
!Digestion
 +
|-
 +
|||DNA (500 ng/ul)||XbaI||SpeI-HF||Cut Smart Buffer||ddH₂O||Total
 +
|-
 +
|#18-2||6.66 ul||0.4 ul||0.4 ul||2.0 ul||8.54 ul||20.0 ul
 +
|-
 +
|#18-5||8.66 ul||0.4 ul||0.4 ul||2.0 ul||10.54 ul||20.0 u
 +
|}
 +
<p>37˚C 40’</p>
 +
 +
<br>
 +
 +
Ligation
 +
{| border="1" class="wikitable"
 +
!Ligation
 +
|-
 +
|||pSB1C3-invF (175 ng/ul)||ColA (100 ng/ul)||T4 DNA Ligase ||T4 DNA Ligase Buffer||ddH₂O||Total
 +
|-
 +
|1||7.0 ul||-||1.0 ul||2.0 ul||9.17 ul||20.0 ul
 +
|-
 +
|2||-||7.0 ul||1.0 ul||2.0 ul||5.0 ul||20.0 ul
 +
|}
 +
<p>Transformation will be made.</p>
 +
 +
<br>
 +
 +
Mini Prep of “pSB1C3 – TEV”
 +
{| border="1" class="wikitable"
 +
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
 +
|-
 +
|1||blank||biospec||9/8/2015 10:39:08 AM||-0.1||ng/ul||-0.003||-0.009||0.30||0.13||DNA||50.00
 +
|-
 +
|2||psb1c3-tev 17||biospec||9/8/2015 10:40:15 AM||125.5||ng/ul||2.510||1.331||1.89||2.16||DNA||50.00
 +
|-
 +
|3||psb1c3-tev 18||biospec||9/8/2015 10:42:45 AM||111.9||ng/ul||2.238||1.195||1.87||2.14||DNA||50.00
 +
|}
 +
 +
<br>
 +
 +
Digestion of “pSB1C3 – TEV 17/18 and pColA-Toehold”
 +
{| border="1" class="wikitable"
 +
!Digestion of “pSB1C3 – TEV 17/18”
 +
|-
 +
|||DNA (1000 ng/ul)||XbaI (FD)||SpeI (FD)||Fast Digest Buffer||ddH₂O||Total
 +
|-
 +
|17||8.0 ul||1.0 ul||1.0 ul||2.0 ul||8.0 ul||20.0 ul
 +
|-
 +
|18||9.0 ul||1.0 ul||1.0 ul||2.0 ul||7.0 ul||20.0 ul
 +
|}
 +
<p>37˚C 1h</p>
 +
 +
<br>
 +
 +
{| border="1" class="wikitable"
 +
!Digestion of “pColA-Toehold”
 +
|-
 +
|||DNA (1000 ng/ul)||XbeI (FD)||SpeI (FD)||Fast Digest Buffer||ddH₂O||Total
 +
|-
 +
|1x||16.0 ul||1.0 ul||1.0 ul||2.0 ul||-||20.0 ul
 +
|}
 +
<p>37˚C 1h</p>
 +
 +
<br>
 +
 +
Colony PCR of “ pSB1C3-TEV 17/18” (08.09.2015)
 +
{| border="1" class="wikitable"
 +
!Colony PCR
 +
|-
 +
|||MgCl₂||(NH₄)2SO₄||pTRE Luc fw||SV40 rv||dNTP||Tag||ddH₂O||DNA||Total
 +
|-
 +
|1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul
 +
|-
 +
|25x||61.5 ul||61.5 ul||25.0 ul||25.0 ul||12.5 ul||5.0 ul||307.5 ul||||498.0 ul
 +
|}
 +
{| border="1" class="wikitable"
 +
!Cycling
 +
|-
 +
|||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle
 +
|-
 +
|Time||5’||30’’||30’’||1.5’||5’||35x
 +
|}
 +
<p>Result: negative</p>
 +
GEL GÖRÜNTÜSÜ
 +
 +
<br>
 +
 +
Digestion of “pColA / pSB1C3 #9 / pColA-Toehold #2-8 / pSB1C3 T7 #10-17 / #18” (10.11.2015)
 +
{| border="1" class="wikitable"
 +
!Digestion of
 +
|-
 +
|||DNA (1000 ng/ul)||XbaI (FD)||SpeI (FD)||Cut Smart Buffer||ddH₂O||Total
 +
|-
 +
|pColA||4.5 ul||0.5 ul||0.5 ul||2.0 ul||12.5 ul||20.0 ul
 +
|-
 +
|pSB1C3 #9||1.7 ul||0.5 ul||0.5 ul||2.0 ul||15.3 ul||20.0 ul
 +
|-
 +
|pColA-Toehold #2-8||16.0 ul||0.5 ul||0.5 ul||2.0 ul||1.0 ul||20.0 ul
 +
|-
 +
|pSB1C3 T7 #10-17||3.3 ul||0.5 ul||0.5 ul||2.0 ul||13.7 ul||20.0 ul
 +
|-
 +
|#18||3.0 ul||0.5 ul||0.5 ul||2.0 ul||14.0 ul||20.0 ul
 +
|}
 +
<p>37˚C 3h<7p>
 +
<p>pColA-Toehold #2-8: 1ul rSAP / 37˚C 1h / 80˚C 20’</p>
 +
<p>Result: pColA and #10 gel extraction will be made.</p>
 +
GEL GÖRÜNTÜLERI
 +
 +
<br>
 +
 +
Gel extraction
 +
{| border="1" class=wikitable"
 +
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
 +
|-
 +
|1||blank||biospec||9/10/2015 7:29:27 PM||-0.2||ng/ul||-0.003||-0.009||0.37||0.28||DNA||50.00
 +
|-
 +
|2||psb1c3-#9||biospec||9/10/2015 7:31:01 PM||308.0||ng/ul||6.160||3.182||1.94||2.10||DNA||50.00
 +
|}
 +
{| border="1" class="wikitable"
 +
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
 +
|-
 +
|1||blank||biospec||9/10/2015 8:13:13 PM||0.3||ng/ul||0.005||-0.003||-1.70||-2.25||DNA||50.00
 +
|-
 +
|2||psb1c3 t7-10 (17)||biospec||9/10/2015 8:14:54 PM||152.2||ng/ul||3.045||1.612||1.89||2.25||DNA||50.00
 +
|-
 +
|3||psb1c3 t7-10 (18)||biospec||9/10/2015 8:16:02 PM||164.6||ng/ul||3.291||1.737||1.90||2.23||DNA||50.00
 +
|}
 +
{| border="1" class="wikitable"
 +
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
 +
|-
 +
|1||blank||biospec||9/11/2015 10:05:05 PM||0.1||ng/ul||0.002||-0.017||-0.13||-0.11||DNA||50.00
 +
|-
 +
|2||pcola (notI)||biospec||9/11/2015 10:07:08 PM||40.4||ng/ul||0.807||0.399||2.03||1.75||DNA||50.00
 +
|-
 +
|3||psb1c3 #9||biospec||9/11/2015 10:08:33 PM||76.0||ng/ul||1.520||0.789||1.93||2.17||DNA||50.00
 +
|-
 +
|4||psb1c3-t7 #10||biospec||9/11/2015 10:09:55 PM||71.5||ng/ul||1.431||0.721||1.99||2.15||DNA||50.00
 +
|-
 +
|5||blank ||biospec||9/11/2015 10:14:04 PM||-0.4||ng/ul||-0.007||-0.020||0.35||0.32||DNA||50.00
 +
|-
 +
|6||psb1c3-t7 #10||biospec||9/11/2015 10:15:01 PM||70.4||ng/ul||1.408||0.727||1.94||2.16||DNA||50.00
 +
|}
 +
{| border="1" class="wikitable"
 +
!#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor
 +
|-
 +
|1||blank||biospec||9/11/2015 2:24:30 AM||0.6||ng/ul||0.011||0.002||4.95||1.18||DNA||50.00
 +
|-
 +
|2||Pcola-2||biospec||9/11/2015 2:26:25 AM||145.0||ng/ul||2.901||1.398||2.08||2.04||DNA||50.00
 +
|}
 +
 +
 +
<html>
 +
 +
 +
</section>
 +
</div>
 +
 
</div>
 
</div>
  

Revision as of 09:32, 18 September 2015


NOTEBOOK

December

9.12.-13.12. We filled a survey to become next generation of ATOMS members.

Waiting in excitement and wondering who will be the members of this year’s team.

22.12. Aaaaand the results of the interviews we made are announced! Now we’re a crowded team of 25 people. Let’s see what’ll happen next.

26.12. Had our first meeting and our first topic was: sponsorship.


January

We had training classes at 08.01-10.01. January. Now it is time to learn what iGEM wants from us.

10.01.-23.01. We made researches about previous iGEM teams, wondering who did what.

23.01.-28.01. Hooray! It’s holiday! Home sweet home.

It’s time to find our own project. After now, we started to have meetings every Monday and Thursday, becase we need to work a lot for finding a new project. Also, still trying to find sponsors.


February

01.02. As we are ATOMS, those Sundays needed to be filled too! We made subgroups in our team and started to search previous Grand Prized-Teams. Now every group will present a team at the meetings.

We have just built ATOMS gene library.

02.02. We decided to attend some competitions due to the lack of sponsors. We attended ‘illnesses with imagery’. Talented people is a must in a team.

13.02. Drawings were done and sent. We believe we will be successful. We are still doing researches in full swing.


March

05.03. Today they filmed our school, and our laboratory so. We had so much fun while they were shooting us!

11.03. The art competiton’s results are just announced. Disappointment. There was not even a winner of first place. We convinced ourselves with thinking they don’t have a sense of art.

14.03. We made a biobrcik design workshop till we see the sunlight! At the late times of night, every group explained their biobrick designs. Now everyone knows what a promoter is :)

17.03. Lab-cleaning party! We don’t wanna be contaminated.


April

05.04. We bid farewell to our teammate Furkan Beştepe. He went to USA.

10.04. Happy birthday Gülnihal! By the way your birthday cake was yummy.

12.04. Daytime wasn’t enough so we started to have meetings in the nights! After working a lot, we deserved eating a delicious meal. Today we also started to search about Toehold.

13.04. We are still learnig about Toehold. It has a potential to be used.

27.04. Fun at the lab.

30.04. ATOMS Ladieas worked on a universal blood idea till the morning.



May

03.05. We must hurry up to find a project. Camp time at Asya Thermal Hotel.

04.05. Why there isn’t anyone in the lab?

06.05. Finally found our project idea! ULCER AND CANCER

09.05. Perfecting our cancer switch system.

15.05. Presented our project to the dean Mehmet Gündüz and the whole genetic department of our faculty! He treated us with Turkish tea and snacks.

24.05. We held a meet-up with other Turkish iGEM teams! Time to make some presentations.


June

This month we designed our genes and studied for our exams at the same time. In short it was a tough week.

Week 1
Gradient PCR of „pCAG with CMV-Enhancer FWD/Cβ-Actin REV. Primers” (29.06.2015)

Gradient PCR from pCAGGS
MgCl₂ (NH₄)2SO₄ VR fwd VR rv dNTP Tag ddH₂O DNA Total
1x 2.5 ul 2.5 ul 0.5 ul 0.5 ul 0.5 ul 0.2 ul 16.3 ul 2.0 ul 25.0 ul
9X 22.5 ul 22.5 ul 4.5 ul 4.5 ul 4.5 ul 1.8 ul 146.7 ul 18.0 ul 225.0 ul

57-64˚C

Results weren’t matching with expected results, experiment will be repeated.


(30.06.2015)

Gradient PCR from pCAGGS
MgCl₂ (NH₄)2SO₄ VR fwd VR rv dNTP Tag ddH₂O DNA Total
1x 2.5 ul 2.5 ul 0.5 ul 0.5 ul 0.5 ul 0.2 ul 16.3 ul 2.0 ul 25.0 ul
9X 22.5 ul 22.5 ul 4.5 ul 4.5 ul 4.5 ul 1.8 ul 146.7 ul 18.0 ul 225.0 ul

52-59˚C

Results weren’t matching with expected results, experiment will be repeated.

August

11.08. We started designing our wiki.

18.08.2015 We got a little tired of doing experiments, so treated ourselves with an awesome team dinner at a fish restaurant.

22.08.2015 A team dinner again, but this time our dear professors Mehmet and Esra Gündüz hosted us at their house.

29.08.2015 Wiki’s tour part is drawn, now it’s time to color these drawings!

Week 6
Mini Prep of “pSB1C3-RFP”

# Sample ID User name Date and Time Nucleic Acid Conc. Unit A260 A280 260/280 260/230 Sample Type Factor
1 blank biospec 8/1/2015 6:03:37 PM -0.1 ng/ul -0.003 -0.009 0.30 0.12 DNA 50.00
2 psb1c3-A biospec 8/1/2015 6:06:00 PM 207.0 ng/ul 4.140 2.184 1.90 2.11 DNA 50.00
3 psb1c3-B biospec 8/1/2015 6:07:07 PM 209.0 ng/ul 4.179 2.210 1.89 2.19 DNA 50.00
4 psb1c3-C biospec 8/1/2015 6:08:05 PM 157.3 ng/ul 3.147 1.664 1.89 2.09 DNA 50.00
5 psb1c3-D biospec 8/1/2015 6:08:58 PM 242.5 ng/ul 4.851 2.548 1.90 2.11 DNA 50.00
6 psb1c3-E biospec 8/1/2015 6:09:42 PM 116.4 ng/ul 2.329 1.215 1.92 2.17 DNA 50.00
7 psb1c3-F biospec 8/1/2015 6:10:28 PM 236.4 ng/ul 4.729 2.503 1.89 2.17 DNA 50.00
8 psb1c3-71 biospec 8/1/2015 6:11:33 PM 111.7 ng/ul 2.233 1.175 1.90 2.10 DNA 50.00
9 psb1c3-72 biospec 8/1/2015 6:12:14 PM 106.4 ng/ul 2.127 1.100 1.93 2.23 DNA 50.00
10 psb1c3-8 biospec 8/1/2015 6:12:51 PM 90.7 ng/ul 1.814 0.959 1.89 1.98 DNA 50.00


Ligation of „pSB1C3 – Gblocks“

2 3 5 6 7 8 9 10 11 12 13 14 15
Insert 7.3 ul 7.0 ul 4.8 ul 8.4 ul 5.8 ul 8.3 ul 3.6 ul 3.9 ul 3.4 ul 4.4 ul 5.9 ul 3.9 ul 5.9 ul
Vector 2.0 ul 2.0 ul 2.0 ul 2.0 ul 2.0 ul 2.0 ul 2.0 ul 2.0 ul 2.0 ul 2.0 ul 2.0 ul 2.0 ul 2.0 ul
Buffer 2.0 ul 2.0 ul 2.0 ul 2.0 ul 2.0 ul 2.0 ul 2.0 ul 2.0 ul 2.0 ul 2.0 ul 2.0 ul 2.0 ul 2.0 ul
Enzyme 1.0 ul 1.0 ul 1.0 ul 1.0 ul 1.0 ul 1.0 ul 1.0 ul 1.0 ul 1.0 ul 1.0 ul 1.0 ul 1.0 ul 1.0 ul
ddH₂O 7.7 ul 8.0 ul 10.2 ul 6.6 ul 9.2 ul 6.7 ul 11.4 ul 11.1 ul 11.6 ul 10.6 ul 9.1 ul 11.1 ul 9.1 ul
Total 20.0 ul 20.0 ul 20.0 ul 20.0 ul 20.0 ul 20.0 ul 20.0 ul 20.0 ul 20.0 ul 20.0 ul 20.0 ul 20.0 ul 20.0 ul


Week 7
Colony PCR of „pSB1C3 – Gblocks“ (03.08.2015)

Colony PCR
MgCl₂ (NH₄)2SO₄ VR fwd VR rv dNTP Tag ddH₂O DNA Total
1x 2.5 ul 2.5 ul 1.0 ul 1.0 ul 0.5 ul 0.2 ul 12.3 ul 5.0 ul 25.0 ul
22X 55.0 ul 55.0 ul 22.0 ul 22.0 ul 11.0 ul 4.4 ul 271.0 ul 440.4 ul
Cycling
95˚C 95˚C 56˚C 72˚C 72˚C Cycle
Time 5’ 30’’ 30’’ 1.5’ 5’ 35x

<a href="ATOMS-Turkiye_03_08_colony_pcr.jpeg" data-lightbox="image-1" class="img="left"><img src="ATOMS-Turkiye_03_08_colony_pcr.jpeg" width="410" height="340"></a>

Result: negative


Colony PCR of „pSB1C3 – Gblocks #14“ (03.08.2015)

Colony PCR
MgCl₂ (NH₄)2SO₄ VR fwd VR rv dNTP Tag ddH₂O DNA Total
1x 2.5 ul 2.5 ul 1.0 ul 1.0 ul 0.5 ul 0.2 ul 12.3 ul 5.0 ul 25.0 ul
36X 90.0 ul 90.0 ul 36.0 ul 36.0 ul 18.0 ul 7.2 ul 442.8 ul 720.0 ul
Cycling
95˚C 95˚C 56˚C 72˚C 72˚C Cycle
Time 5’ 30’’ 30’’ 1.5’ 5’ 35x

<a href="ATOMS-Turkiye_03_08_colony_pcr_14.jpeg" data-lightbox="image-1" class="img="left"><img src="ATOMS-Turkiye_03_08_colony_pcr_14.jpeg" width="410" height="360"></a> <a href="ATOMS-Turkiye_03_08_colony_pcr_14_1.jpeg" data-lightbox="image-1" class="img="left"><img src="ATOMS-Turkiye_03_08_colony_pcr_14_1.jpeg" width="360" height="360"></a>

Result: negative


Digestion of “pSB1C3-RFP” (03.08.2015)

Digestion of “pSB1C3-RFP”
Vector Fast Digest Buffer EcoRI (FD) PstI (FD) ddH₂O Total
1x 2.0 ul 2.0 ul 1.0 ul 1.0 ul 14.0 ul 20.0 ul

37˚C 2h

<a href="ATOMS-Turkiye_03_08_rfp_kesim.jpeg" data-lightbox="image-1" class="img="left"><img src="ATOMS-Turkiye_03_08_rfp_kesim.jpeg" width="410" height="360"></a>

Gel Electropheresis was made.

Result: Bands were at the expected section. Gel purification was made.



Ligation of “pSB1C3 – Gblock #14”

Ligation of “pSB1C3 – Gblock #14”
Insert Vector T4 DNA Ligase Buffer T4 DNA Ligase ddH₂O Total
1) PCR clean up vektörü ile 2.5 ul 2.5 ul 2.0 ul
(100ng) 2.0 ul
1.0 ul 12.5 ul 20.0 ul
2) Gel Extraction 7.5 ul 7.5 ul 0.8 ul
(50ng) 2.0 ul
1.0 ul 8.7 ul 20.0 ul


# Sample ID User name Date and Time Nucleic Acid Conc. Unit A260 A280 260/280 260/230 Sample Type Factor
1 blank biospec 8/3/2015 6:10:02 PM 0.2 ng/ul 0.004 -0.010 -0.41 1.13 DNA 50.00
2 psb1c3 gel biospec 8/3/2015 6:12:16 PM 62.5 ng/ul 1.250 0.664 1.88 0.90 DNA 50.00
4 psb1c3 clean-up biospec 8/3/2015 6:13:39 PM 170.5 ng/ul 3.409 1.884 1.81 1.33 DNA 50.00


PCR of “G-Bloks via Q5 Polymerase” (04.08.2015)

PCR
SV40 poly A rev CMV fwd tetR rev 2x Q5 Master Mix DNA Total
1x 2.5 ul 2.5 ul 2.5 ul 10.0 ul 5.0 ul 20.0 ul


Cycling SV40 rev/CMV fwd
95˚C 95˚C 63˚C 72˚C 72˚C Cycle
Time 5’ 30’’ 30’’ 1’ 2’ 35x


Cycling CMV fwd/tetR rev
98˚C 98˚C 66˚C 72˚C 72˚C Cycle
Time 30’’ 10’’ 30’’ 1’ 2’ 35x


Digestion of “pSB1C3-RFP” (04.08.2015)

Digestion
Vector Cut Smart Buffer EcoRI SpeI ddH₂O Total
1x 2.0 ul 2.0 ul 1.0 ul 1.0 ul 14.0 ul 20.0 ul

37˚C 4h

<a href="ATOMS-Turkiye_04_08_15.jpeg" data-lightbox="image-1" class="img="left"><img src="ATOMS-Turkiye_04_08_15.jpeg" width="410" height="360"></a>

Result: Bands were at the expected section.


# Sample ID User name Date and Time Nucleic Acid Conc. Unit A260 A280 260/280 260/230 Sample Type Factor
1 blank biospec 8/4/2015 7:03:09 AM 0.9 ng/ul 0.017 0.005 3.41 0.68 DNA 50.00
2 Blank2 biospec 8/4/2015 7:04:51 AM -0.5 ng/ul -0.010 -0.017 0.59 0.45 DNA 50.00
4 clean up biospec 8/4/2015 7:05:55 AM 86.8 ng/ul 1.735 0.913 1.90 0.40 DNA 50.00
5 gel ext biospec 8/4/2015 7:06:51 AM 36.2 ng/ul 0.723 0.380 1.90 0.29 DNA 50.00


Colony PCR of “11,12,13 pSB1C3 - GBlocks”

Colony PCR
MgCl₂ (NH₄)2SO₄ VF VR Tag ddH₂O DNA Total
1x 2.5 ul 2.5 ul 0.5 ul 0.5 ul 0.2 ul 13.3 ul 5.0 ul 25.0 ul
17X 42.5 ul 42.5 ul 8.5 ul 8.5 ul 3.4 ul 226.1 ul 340.0 ul
Cycling CMV fwd/tetR rev
95˚C 95˚C 56˚C 72˚C 72˚C Cycle
Time 5’ 30’’ 30’’ 1.5’ 5’ 35x

<a href="ATOMS-Turkiye_04_08_15_colony_pcr_11%2C12%2C13.jpeg" data-lightbox="image-1" class="img="left"><img src="ATOMS-Turkiye_04_08_15_colony_pcr_11%2C12%2C13.jpeg" width="410" height="360"></a>

Result: negative


Ligation and Transformation of “5,6,7,8,9 pSB1C3-GBlocks”

Ligation of “GBlocks 11-12 pSB1C3”
Vector Gen Buffer Ligase ddH₂O Total
5 0.8 ul 2.37 ul 1.0 ul 0.5 ul 5.33 ul 10.0 ul
6 0.8 ul 4.2 ul 1.0 ul 0.5 ul 3.5 ul 10.0 ul
7 0.8 ul 2.94 ul 1.0 ul 0.5 ul 4.76 ul 10.0 ul
8 0.8 ul 4.16 ul 1.0 ul 0.5 ul 3.54 ul 10.0 ul
9 0.8 ul 1.79 ul 1.0 ul 0.5 ul 5.91 ul 10.0 ul

50ng DNA

1 vector:0.5 insert

Transfomation with TOP10.


Ligation of “GBlocks 11-12 pSB1C3”

Ligation of “GBlocks 11-12 pSB1C3”
pSB1C3 Insert Buffer Ligase ddH₂O Total
11 0.8 ul 1.7 ul 1.0 ul 0.5 ul 6.0 ul 10.0 ul
12 0.4 ul 1.1 ul 1.0 ul 0.5 ul 7.0 ul 10.0 ul

Transformation was made.


Colony PCR of “5,6,7,8,9,T7 system – Gblocks”

Colony PCR
MgCl₂ (NH₄)2SO₄ VR VF dNTP Tag ddH₂O DNA Total
1x 2.5 ul 2.5 ul 0.5 ul 0.5 ul 0.5 ul 0.1 ul 13.3 ul 5.0 ul 24.9 ul
58X 145.0 ul 145.0 ul 29.0 ul 29.0 ul 29.0 ul 5.8 ul 771.4 ul 1154.2 ul
Cycling
95˚C 95˚C 56˚C 72˚C 72˚C Cycle
Time 5’ 30’’ 30’’ 1.5’ 5’ 35x


Ligation of “GBlocks 2,5,6,7,8,9,10,12,13,15,17 pSB1C3-RFP”

Ligation of “GBlocks 11-12 pSB1C3”
pSB1C3 Insert Buffer Ligase ddH₂O Total
2 1.4 ul 3.65 ul 1.0 ul 0.5 ul 3.45 ul 10.0 ul
5 1.4 ul 2.4 ul 1.0 ul 0.5 ul 4.7 ul 10.0 ul
6 1.4 ul 4.2 ul 1.0 ul 0.5 ul 2.9 ul 10.0 ul
7 1.4 ul 2.9 ul 1.0 ul 0.5 ul 4.2 ul 10.0 ul
8 1.4 ul 4.15 ul 1.0 ul 0.5 ul 2.95 ul 10.0 ul
9 1.4 ul 1.8 ul 1.0 ul 0.5 ul 5.3 ul 10.0 ul
10 1.4 ul 1.9 ul 1.0 ul 0.5 ul 5.2 ul 10.0 ul
12 1.4 ul 2.2 ul 1.0 ul 0.5 ul 4.9 ul 10.0 ul
13 1.4 ul 2.9 ul 1.0 ul 0.5 ul 4.2 ul 10.0 ul
15 1.4 ul 2.95 ul 1.0 ul 0.5 ul 4.15 ul 10.0 ul
17 1.4 ul 1.25 ul 1.0 ul 0.5 ul 5.75 ul 10.0 ul

Transformation was made.



Colony PCR of “pTRE-1,13,15” (06.08.2015)

Colony PCR
MgCl₂ (NH₄)2SO₄ CMV fwd SV40 rv dNTP Tag ddH₂O DNA Total
1x 2.5 ul 2.5 ul 1.0 ul 1.0 ul 0.5 ul 0.2 ul 12.3 ul 5.0 ul 25.0 ul
20X 50.0 ul 50.0 ul 20.0 ul 20.0 ul 10.0 ul 2.0 ul 246.0 ul 400.0 ul
Cycling
95˚C 95˚C 56˚C 72˚C 72˚C Cycle
Time 5’ 30’’ 30’’ 1.5’ 5’ 35x

<a href="ATOMS-Turkiye_07_08_colony_pcr_1%2C2%2C3.jpeg" data-lightbox="image-1" class="img="left"><img src="ATOMS-Turkiye_07_08_colony_pcr_1%2C2%2C3.jpeg" width="410" height="360"></a>

Result: negative

GEL GÖRÜNTÜSÜ


Colony PCR of “5,6,7,9 – Gblocks” (06.08.2015)

Colony PCR
MgCl₂ (NH₄)2SO₄ VR VF dNTP Tag ddH₂O DNA Total
1x 2.5 ul 2.5 ul 0.5 ul 0.5 ul 0.5 ul 0.2 ul 13.3 ul 5.0 ul 25.0 ul
40X 100.0 ul 100.0 ul 20.0 ul 20.0 ul 20.0 ul 8.0 ul 532.0 ul 800.0 ul
Cycling
95˚C 95˚C 56˚C 72˚C 72˚C Cycle
Time 5’ 30’’ 30’’ 1.5’ 5’ 35x

Result: negative

GEL GÖRÜNTÜSÜ


Colony PCR of “7,8,10 – Gblocks” from Masterplate (08.08.2015)

Colony PCR
MgCl₂ (NH₄)2SO₄ T7 terminatör Rev. T7 terminatör For. dNTP Tag ddH₂O DNA Totalzz
1x 2.5 ul 2.5 ul 1.0 ul 1.0 ul 0.5 ul 0.5 ul 12.3 ul 5.0 ul 25.0 ul
26X 65.0 ul 65.0 ul 26.0 ul 26.0 ul 13.0 ul 13.0 ul 319.8 ul 527.8 ul
Cycling
95˚C 95˚C 56˚C 72˚C 72˚C Cycle
Time 5’ 30’’ 30’’ 1.5’ 5’ 35x

Result: negative

GEL GÖRÜNTÜSÜ


Digestion of “pET45” (09.08.2015)

Digestion
pET45 (455ng/ul) pSB1C3-RFP (400 ng/ul) Cut Smart Buffer XhoI (Neb) BamHI-HF (Neb) EcoRI-HF (Neb) SpeI (Neb) ddH₂O Total
1 - 5.0 ul 2.0 ul - - 1.0 ul 1.0 ul 11.0 ul 20.0 ul
2 4.0 ul - 2.0 ul 1.0 ul 1.0 ul - - 12.0 ul 20.0 ul

37˚C 3.5h

Result: Positive. Gel extraction was made.

GEL GÖRÜNTÜSÜ

# Sample ID User name Date and Time Nucleic Acid Conc. Unit A260 A280 260/280 260/230 Sample Type Factor
1 blank biospec 8/9/2015 5:04:14 PM -0.6 ng/ul -0.011 0.005 3.41 0.68 DNA 50.00
2 pet45 x+b biospec 8/9/2015 5:05:26 PM 106.9 ng/ul 2.138 -0.017 0.59 0.45 DNA 50.00
4 psb1c3 e+s biospec 8/9/2015 5:06:20 PM 133.3 ng/ul 2.666 0.913 1.90 0.40 DNA 50.00


Ligation of “PET45 – HNS” (10.08.2015)

Ligation
Vector Insert T4 DNA Ligase Buffer T4 DNA Ligase ddH₂O Total
1x 2.1 ul 1.0 ul 2.0 ul 0.5 ul 14.4 ul 20.0 ul

16˚C 2h


Digestion of “pSB1C3 – HNS”

Ligation
Gen BamHI XhoI Cut Smart Buffer ddH₂O Total
1x 11.5 ul 1.0 ul 1.0 ul 2.0 ul 4.5 ul 20.0 ul

37˚C 3h


Colony PCR of “pTRE #13”

Colony PCR
MgCl₂ (NH₄)2SO₄ CMV for. SV40 rev. dNTP Tag ddH₂O DNA Total
1x 2.5 ul 2.5 ul 1.0 ul 1.0 ul 0.5 ul 0.2 ul 12.3 ul 5.0 ul 25.0 ul
10X 25.0 ul 25.0 ul 10.0 ul 10.0 ul 5.0 ul 2.0 ul 123.0 ul 250.0 ul
Cycling
95˚C 95˚C 60˚C 72˚C 72˚C Cycle
Time 5’ 30’’ 30’’ 1.5’ 5’ 35x

Result: negative

GEL GÖRÜNTÜSÜ


Colony PCR of “PET45-4,5,6,7,8,9,10” (10.08.2015)

Colony PCR
MgCl₂ (NH₄)2SO₄ T7 ter. fw T7 ter. rev dNTP Tag ddH₂O DNA Total
1x 2.5 ul 2.5 ul 1.0 ul 1.0 ul 0.5 ul 0.2 ul 12.3 ul 5.0 ul 25.0 ul
86X 215.0 ul 215.0 ul 86.0 ul 186.0 ul 43.0 ul 17.2 ul 1075.0 ul 1720.0 ul
Cycling
95˚C 95˚C 56˚C 72˚C 72˚C Cycle
Time 5’ 30’’ 30’’ 1.5’ 5’ 35x

Result: Bands were at the expected section. Liquid Culture was made.

GEL GÖRÜNTÜLERI


Digestion of „pColA-KanR and „pSB1C3-Toehold“ (10.08.2015)

Digestion
pColA-KanR (500 ng/ul) pSB1C3-Toehold (500 ng/ul) NotI-HF (Neb) Cut Smart Buffer ddH₂O Total
1 11.6 ul - 1.0 ul 2.0 ul 5.4 ul 20.0 ul
2 - 6.5 ul 1.0 ul 2.0 ul 10.5 ul 20.0 ul

37˚C 2h

pColA-KanR: 1.0 ul rSAP/37˚C 1h/-80˚C 20’’ heat shock

GEL GÖRÜNTÜSÜ


Ligation of “pColA-KanR and pSB1C3-Toehold”

Ligation
pColA-KanR (500 ng/ul) pSb1C3-Toehold T4 DNA Ligase Buffer (Thermo) T4 DNA Ligase (Thermo) ddH₂O Total Vector:Insert Ratios
1 2.0 ul 1.0 ul 2.0 ul 1.0 ul 14.0 ul 20.0 ul 1:0.33
2 2.0 ul 3.0 ul 2.0 ul 1.0 ul 12.0 ul 20.0 ul 1:1

22˚C 1h


Mini Prep of “pColA-3,4,5”

# Sample ID User name Date and Time Nucleic Acid Conc. Unit A260 A280 260/280 260/230 Sample Type Factor
1 blank biospec 8/11/2015 11:11:42 PM -0.2 ng/ul -0.003 -0.007 0.49 0.13 DNA 50.00
2 ColA-3 biospec 8/11/2015 11:13:07 PM 27.2 ng/ul 0.544 0.260 2.10 1.95 DNA 50.00
3 ColA-4 biospec 8/11/2015 11:14:38 PM 25.5 ng/ul 0.509 0.253 2.01 1.82 DNA 50.00
4 ColA-4(2) biospec 8/11/2015 11:15:58 PM 25.8 ng/ul 0.517 0.255 2.022 1.81 DNA 50.00
5 ColA-5 biospec 8/11/2015 11:17:02 PM 44.4 ng/ul 0.887 0.449 1.98 1.76 DNA 50.00

Result: The Band of pColA were at the expected section. 2000 pb

(CUT CHECK GÖRÜNTÜSÜ)(12.08.2015)


Ligation of “#2 GBlock with pET45” (11.08.2015)

Ligation
pET45 (100 ng/ul) #2 GBlock T4 DNA Ligase Buffer T4 DNA Ligase ddH₂O Total
1x 1.0 ul 7.3 ul 2.0 ul 1.0 ul 8.7 ul 20.0 ul

22˚C - 1’40’’

Transformation was made.


Mini Prep

# Sample ID User name Date and Time Nucleic Acid Conc. Unit A260 A280 260/280 260/230 Sample Type Factor
1 blank biospec 8/12/2015 7:38:28 PM 0.4 ng/ul 0.008 -0.001 -8.91 0.76 DNA 50.00
2 pet45 4-4 biospec 8/12/2015 7:40:21 PM 40.3 ng/ul 0.807 0.402 2.01 2.44 DNA 50.00
3 pet45 4-3 biospec 8/12/2015 7:41:10 PM 53.1 ng/ul 1.063 0.570 1.86 1.74 DNA 50.00
4 pet45 8-12 biospec 8/12/2015 7:41:55 PM 61.8 ng/ul 1.236 0.642 1.92 2.08 DNA 50.00
5 pet45 8-3 biospec 8/12/2015 7:42:37 PM 32.9 ng/ul 0.658 0.332 1.98 2.00 DNA 50.00
6 pet45 9-11 biospec 8/12/2015 7:44:03 PM 34.5 ng/ul 0.690 0.345 2.00 1.77 DNA 50.00
7 pet45 8-1 biospec 8/12/2015 7:44:54 PM 39.0 ng/ul 0.780 0.408 1.91 1.72 DNA 50.00
8 pet45 5-10 biospec 8/12/2015 7:45:49 PM 43.2 ng/ul 0.865 0.441 1.96 1.81 DNA 50.00
9 pet45 6-3 biospec 8/12/2015 7:46:42 PM 43.5 ng/ul 0.870 0.437 1.99 1.92 DNA 50.00
10 pet45 9-8 biospec 8/12/2015 7:47:23 PM 48.1 ng/ul 0.961 0.496 1.94 1.88 DNA 50.00
11 pet45 7-4 biospec 8/12/2015 7:48:06 PM 36.2 ng/ul 0.723 0.372 1.94 1.97 DNA 50.00
12 pet45 4-1 biospec 8/12/2015 7:48:50 PM 49.4 ng/ul 0.987 0.509 1.94 1.99 DNA 50.00
13 pet45 4-2 biospec 8/12/2015 7:49:43 PM 59.3 ng/ul 1.187 0.606 1.96 2.09 DNA 50.00
14 pet45 7-3 biospec 8/12/2015 7:50:33 PM 30.2 ng/ul 0.604 0.307 1.97 1.96 DNA 50.00
15 psb1c3 12 biospec 8/12/2015 7:51:18 PM 141.1 ng/ul 2.823 1.462 1.93 2.14 DNA 50.00
16 pet45 5-7 biospec 8/12/2015 7:52:04 PM 26.1 ng/ul 0.522 0.265 1.97 2.00 DNA 50.00
17 pet45 5-3 biospec 8/12/2015 7:52:49 PM 46.1 ng/ul 0.923 0.470 1.96 2.02 DNA 50.00
18 pet45 6-12 biospec 8/12/2015 7:53:36 PM 29.3 ng/ul 0.586 0.283 2.07 1.80 DNA 50.00
19 pet45 1-12 biospec 8/12/2015 7:54:25 PM 21.4 ng/ul 0.428 0.210 2.04 1.72 DNA 50.00
20 pet45 5-8 biospec 8/12/2015 7:55:08 PM 29.1 ng/ul 0.582 0.299 1.95 2.07 DNA 50.00
21 psb1c3 1 biospec 8/12/2015 7:55:51 PM 75.3 ng/ul 1.507 0.779 1.93 2.07 DNA 50.00
22 pet45 8-4 biospec 8/12/2015 7:56:33 PM 44.1 ng/ul 0.882 0.451 1.96 1.99 DNA 50.00
23 pet45 7-2 biospec 8/12/2015 7:57:16 PM 25.3 ng/ul 0.506 0.259 1.95 1.70 DNA 50.00


# Sample ID User name Date and Time Nucleic Acid Conc. Unit A260 A280 260/280 260/230 Sample Type Factor
1 blank biospec 8/12/2015 7:17:01 PM -0.3 ng/ul -0.007 -0.015 0.47 0.23 DNA 50.00
2 psb1c3-4hns biospec 8/12/2015 7:18:16 PM 61.9 ng/ul 1.237 0.652 1.90 2.17 DNA 50.00
3 pet45 10-1 biospec 8/12/2015 7:19:32 PM 46.7 ng/ul 0.934 0.486 1.92 2.00 DNA 50.00
4 pet45 10-6 biospec 8/12/2015 7:20:23 PM 52.0 ng/ul 1.040 0.549 1.89 1.88 DNA 50.00
5 pet45 10-3 biospec 8/12/2015 7:21:14 PM 51.9 ng/ul 1.037 0.536 1.94 2.10 DNA 50.00
6 pet45 9-6 biospec 8/12/2015 7:22:10 PM 37.7 ng/ul 0.755 0.389 1.94 1.99 DNA 50.00
7 pet45 10-8 biospec 8/12/2015 7:23:08 PM 85.3 ng/ul 1.705 1.002 1.70 0.93 DNA 50.00
8 psb1c3 16-trigger rna biospec 8/12/2015 7:24:27 PM 125.7 ng/ul 2.514 1.401 1.79 1.32 DNA 50.00
9 pet45 10 biospec 8/12/2015 7:25:23 PM 84.7 ng/ul 1.694 1.025 1.65 0.76 DNA 50.00
10 pet45 6-6 biospec 8/12/2015 7:26:23 PM 100.3 ng/ul 2.006 1.124 1.79 1.24 DNA 50.00
11 pet45 8-2 biospec 8/12/2015 7:27:20 PM 67.5 ng/ul 1.349 0.778 1.73 0.91 DNA 50.00
12 pet45 7-1 biospec 8/12/2015 7:28:07 PM 52.5 ng/ul 1.050 0.549 1.91 1.83 DNA 50.00


Cut Check of “pET45 1,4,5,6,7,8,9,10 – BamHI/XhoI” (13.08.2015)

Ligation
Gen (250 ng/ul) Cut Smart Buffer BamHI (NEB) XhoI (NEB) ddH₂O Total
1 11.7 ul 2.0 ul 1.0 ul 1.0 ul 4.3 ul 20.0 ul
4 6.2 ul 2.0 ul 1.0 ul 1.0 ul 9.8 ul 20.0 ul
5 8.6 ul 2.0 ul 1.0 ul 1.0 ul 7.4 ul 20.0 ul
6 8.6 ul 2.0 ul 1.0 ul 1.0 ul 7.4 ul 20.0 ul
7 8.3 ul 2.0 ul 1.0 ul 1.0 ul 7.7 ul 20.0 ul
8 7.4 ul 2.0 ul 1.0 ul 1.0 ul 8.6 ul 20.0 ul
9 6.7 ul 2.0 ul 1.0 ul 1.0 ul 9.3 ul 20.0 ul
10 5.9 ul 2.0 ul 1.0 ul 1.0 ul 10.1 ul 20.0 ul

Dogru cikan koloniler BL21 bakterisine transformasyon edildi ve sivi kültür yapildi. Sonrasinda protein izolasyon ve WB yapilacak.

GEL GÖRÜNTÜLERI


# Sample ID User name Date and Time Nucleic Acid Conc. Unit A260 A280 260/280 260/230 Sample Type Factor
1 blank biospec 8/13/2015 1:31:59 AM 0.0 ng/ul -0.001 -0.005 0.17 0.08 DNA 50.00
2 psb1c3-e+p biospec 8/13/2015 1:32:58 AM 58.6 ng/ul 1.172 0.618 1.90 0.57 DNA 50.00


Colony PCR of “pTRE – 12,13 GBlocks” (14.08.2015)

Colony PCR
MgCl₂ (NH₄)2SO₄ CMV fw SV40 rv dNTP Tag ddH₂O DNA Total
1x 2.5 ul 2.5 ul 1.0 ul 1.0 ul 0.5 ul 0.2 ul 12.3 ul 5.0 ul 25.0 ul
15X 37.5 ul 37.5 ul 15.0 ul 15.0 ul 7.5 ul 3.0 ul 184.5 ul 300.0 ul
Cycling
95˚C 95˚C 56˚C 72˚C 72˚C Cycle
Time 5’ 30’’ 30’’ 1.5’ 5’ 35x

Sonuc: negative

GEL GÖRÜNTÜSÜ


Colony PCR of “pColA – Toehold GFP” (14.08.2015)

Colony PCR
MgCl₂ (NH₄)2SO₄ pColA fw pColA rv dNTP Tag ddH₂O DNA Total
1x 2.5 ul 2.5 ul 1.0 ul 1.0 ul 0.5 ul 0.2 ul 12.3 ul 5.0 ul 25.0 ul
26X 65.0 ul 65.0 ul 26.0 ul 26.0 ul 13.0 ul 5.2 ul 319.8 ul 520.0 ul
Cycling
95˚C 95˚C 56˚C 72˚C 72˚C Cycle
Time 5’ 30’’ 30’’ 1.5’ 5’ 35x

Result: 2-4, 2-8, 1-6 Bands were at the expected section.


Digestion of “pSB1C3-RFP” (14.08.2015)

Digestion
pSB1C3-RFP (400 ng/ul) Cut Smart Buffer EcoRI-HF PstI ddH₂O Total
1 5.0 ul 2.0 ul 1.0 ul 1.0 ul 11.0 ul 20.0 ul
2 5.0 ul 2.0 ul 1.0 ul 1.0 ul 11.0 ul 20.0 ul
3 5.0 ul 2.0 ul 1.0 ul 1.0 ul 11.0 ul 20.0 ul

37˚C – 3.5h

Gel extraction was made.


Ligation of “pSB1C3-GBlocks”

Ligation
Digested pSB1C3 GBlocks T4 DNA Ligase Buffer T4 DNA Ligase ddH₂O Total
1 1.7 ul 2.0 ul 2.0 ul 1.0 ul 13.3 ul 20.0 ul

22˚C – 1.30h

Transformation with Neb10-β. Colony PCR will be made.


Colony PCR of “GBlocks – 2,5,6,7,8,9” (14.08.2015)

Colony PCR
MgCl₂ (NH₄)2SO₄ VR VF dNTP Tag ddH₂O DNA Total
1x 2.5 ul 2.5 ul 1.0 ul 1.0 ul 0.5 ul 0.2 ul 12.3 ul 5.0 ul 25.0 ul
45X 112.5 ul 112.5 ul 45.0 ul 45.0 ul 22.5 ul 9.0 ul 553.5 ul 900.0 ul
55X 137.5 ul 137.5 ul 55.0 ul 55.0 ul 27.5 ul 11.0 ul 676.5 ul 1100.0 ul
Cycling
95˚C 95˚C 56˚C 72˚C 72˚C Cycle
Time 5’ 30’’ 30’’ 1.5’ 5’ 35x

GEL GÖRÜNTÜLERI


Digestion/Gel Electrophoresis/Gel Extraction and Purification of “pSB1C3” (16.08.2015)

Digestion
DNA FD EcoRI PstI ddH₂O Total
1x 2.5 ul 2.0 ul 1.0 ul 1.0 ul 13.5 ul 20.0 ul

37˚C 1h


# Sample ID User name Date and Time Nucleic Acid Conc. Unit A260 A280 260/280 260/230 Sample Type Factor
1 blank biospec 8/16/2015 10:45:30 PM 0.1 ng/ul 0.003 -0.017 0.17 -0.14 DNA 50.00
2 psb1c3-e+p biospec 8/16/2015 10:47:18 PM 14.0 ng/ul 0.279 0.145 1.92 0.15 DNA 50.00


Colony PCR of “pSB1C3 7,10,12,13 and pET45 2” (14.08.2015)

Colony PCR of “pSB1C3 7,10,12,13”
MgCl₂ (NH₄)2SO₄ VR VF dNTP Tag ddH₂O DNA Total
1x 2.5 ul 2.5 ul 1.0 ul 1.0 ul 0.5 ul 0.2 ul 12.3 ul 5.0 ul 25.0 ul
6X 15.0 ul 15.0 ul 6.0 ul 6.0 ul 3.0 ul 1.2 ul 73.8 ul 120.0 ul
Cycling
95˚C 95˚C 56˚C 72˚C 72˚C Cycle
Time 5’ 30’’ 30’’ 1.5’ 5’ 35x

Result: negative

GEL GÖRÜNTÜSÜ


Colony PCR of “pET45 2”
MgCl₂ (NH₄)2SO₄ VR VF dNTP Tag ddH₂O DNA Total
1x 2.5 ul 2.5 ul 1.0 ul 1.0 ul 0.5 ul 0.2 ul 12.3 ul 5.0 ul 25.0 ul
12X 30.0 ul 30.0 ul 12.0 ul 12.0 ul 6.0 ul 2.4 ul 147.6 ul 240.0 ul
Cycling
95˚C 95˚C 56˚C 72˚C 72˚C Cycle
Time 5’ 30’’ 30’’ 1.5’ 5’ 35x

Result: negative

GEL GÖRÜNTÜSÜ


# Sample ID User name Date and Time Nucleic Acid Conc. Unit A260 A280 260/280 260/230 Sample Type Factor
1 blank biospec 8/16/2015 8:54:05 PM -0.2 ng/ul -0.004 -0.017 0.25 0.12 DNA 50.00
2 psb1c3 13-15 biospec 8/16/2015 8:55:29 PM 125.2 ng/ul 2.505 1.339 1.87 2.01 DNA 50.00
3 psb1c3 15-19 biospec 8/16/2015 8:56:24 PM 93.5 ng/ul 1.871 0.999 1.87 1.94 DNA 50.00
4 psb1c3 7-16 biospec 8/16/2015 8:57:33 PM 149.4 ng/ul 2.988 1.575 1.90 2.14 DNA 50.00
5 psb1c3 10-16 biospec 8/16/2015 8:58:18 PM 138.0 ng/ul 2.760 1.469 1.88 2.13 DNA 50.00
6 psb1c3 10-17 biospec 8/16/2015 8:59:03 PM 185.5 ng/ul 3.710 1.994 1.86 1.86 DNA 50.00
7 psb1c3 7-11 biospec 8/16/2015 8:00:54 PM 73.8 ng/ul 1.475 0.843 1.75 1.08 DNA 50.00
8 psb1c3 13-14 biospec 8/16/2015 8:01:53 PM 178.7 ng/ul 3.575 1.922 1.86 1.87 DNA 50.00
9 psb1c3 7-20 biospec 8/16/2015 8:02:54 PM 73.6 ng/ul 1.472 0.774 1.90 2.05 DNA 50.00
10 pcola toe 2-4 biospec 8/16/2015 8:03:57 PM 40.1 ng/ul 0.801 0.441 1.82 1.13 DNA 50.00
11 pcola toe 2-8 biospec 8/16/2015 8:05:31 PM 48.7 ng/ul 0.974 0.503 1.94 1.61 DNA 50.00
12 pcola toe 1-6 biospec 8/16/2015 8:06:09 PM 54.9 ng/ul 1.098 0.570 1.93 1.62 DNA 50.00
13 pcola blunt 1 biospec 8/16/2015 8:06:52 PM 23.9 ng/ul 0.479 0.244 1.96 1.24 DNA 50.00
14 pcola blunt 6 biospec 8/16/2015 8:07:30 PM 31.1 ng/ul 0.623 0.314 1.98 1.67 DNA 50.00



Ligation of “pColA and pSB1C3 #9 (Damp-Pex)” (18.08.2015)

Ligation
pColA (Vector 50 ng/ul) Insert (Damp-Pex) T4 DNA Ligase Buffer T4 DNA Ligase ddH₂O Total Vector:Insert Ratio
9.1 2.0 ul 1.0 ul 2.0 ul 1.0 ul 14.0 ul 20.0 ul 1:0.33
9.2 2.0 ul 2.8 ul 2.0 ul 1.0 ul 12.2 ul 20.0 ul 1:1


Miniprep of “pTet-off DH5α /Top10/NEB5fα/NEB10β”

# Sample ID User name Date and Time Nucleic Acid Conc. Unit A260 A280 260/280 260/230 Sample Type Factor
1 blank biospec 8/18/2015 3:04:27 PM 0.4 ng/ul 0.008 -0.008 -1.01 1.01 DNA 50.00
2 top10 biospec 8/18/2015 3:05:19 PM 258.5 ng/ul 5.169 2.753 1.88 2.11 DNA 50.00
3 neb5a biospec 8/18/2015 3:06:25 PM 30.6 ng/ul 0.612 0.324 1.89 1.34 DNA 50.00
4 neb5a biospec 8/18/2015 3:07:30 PM 33.4 ng/ul 0.668 0.350 1.91 1.27 DNA 50.00
5 neb10beta biospec 8/18/2015 3:08:22 PM 214.6 ng/ul 4.292 2.264 1.90 2.19 DNA 50.00


Colony PCR of “pET45 #2.8” (15.08.2015)

Colony PCR
MgCl₂ (NH₄)2SO₄ VR VF dNTP Tag ddH₂O DNA Total
1x 2.5 ul 2.5 ul 1.0 ul 1.0 ul 0.5 ul 0.2 ul 12.3 ul 5.0 ul 25.0 ul
25X 62.5 ul 62.5 ul 25.0 ul 25.0 ul 12.5 ul 5.0 ul 307.5 ul 500.0 ul
Cycling
95˚C 95˚C 56˚C 72˚C 72˚C Cycle
Time 5’ 30’’ 30’’ 1.5’ 5’ 35x

Result: negative

GEL GÖRÜNTÜSÜ


Colony PCR of “pSB1C3 17,6,8” (S. 118/15.08.2015)

Colony PCR of “pSB1C3 17,6,8”
MgCl₂ (NH₄)2SO₄ VR VF dNTP Tag ddH₂O DNA Total
1x 2.5 ul 2.5 ul 1.0 ul 1.0 ul 0.5 ul 0.2 ul 12.3 ul 5.0 ul 25.0 ul
32X 80.0 ul 80.0 ul 32.0 ul 32.0 ul 16.0 ul 6.4 ul 393.6 ul 640.0 ul
Cycling
95˚C 95˚C 56˚C 72˚C 72˚C Cycle
Time 5’ 30’’ 30’’ 1.5’ 5’ 35x

Result: 6: Liquid Culture was made.

8: sequencing will be done

17: Colony PCR will be repeated.

GEL GÖRÜNTÜSÜ


Colony PCR of “pColA blunt”

Colony PCR of “pColA blunt”
MgCl₂ (NH₄)2SO₄ ColA fw ColA rev dNTP Tag ddH₂O DNA Total
1x 2.5 ul 2.5 ul 1.0 ul 1.0 ul 0.5 ul 0.2 ul 12.3 ul 5.0 ul 25.0 ul
20X 50.0 ul 50.0 ul 20.0 ul 20.0 ul 10.0 ul 4.0 ul 246.0 ul 400.0 ul
Cycling
95˚C 95˚C 56˚C 72˚C 72˚C Cycle
Time 5’ 30’’ 30’’ 1.5’ 5’ 35x

Result: negative

GEL GÖRÜNTÜSÜ


PCR of “Q5 DNA Polymerase for pCAG” (19.08.2015)

PCR
Template DNA pCAG fw pCAG rev Master 2x Mix ddH₂O Total
1x 1.0 ul 5.0 ul 5.0 ul 25.0 ul 14.0 ul 50.0 ul
Cycling
98˚C 98˚C 70˚C 72˚C 72˚C Cycle
Time 30’’ 10’’ 30’’ 1’ 5’ 35x

Result: Bands were at the expected section. PCR clean up will be made.

GEL GÖRÜNTÜSÜ


Digestion of “pCAG – Clean up”

Digestion
pCAG – Clean up (34 ng/ul) pCAG – Clean up (61.6 ng/ul) EcoRI-HF (NEB) XhoI (NEB) Cut Smart Buffer ddH₂O Total
1 7.4 ul - 0.5 ul 0.5 ul 2.0 ul 9.6 ul 20.0 ul
2 - 4.1 ul 0.5 ul 0.5 ul 2.0 ul 12.9 ul 20.0 ul

37˚C overnight


# Sample ID User name Date and Time Nucleic Acid Conc. Unit A260 A280 260/280 260/230 Sample Type Factor
1 blank biospec 8/19/2015 7:08:39 PM 0.0 ng/ul 0.000 0.000 27.59 -0.07 DNA 50.00
2 pcag biospec 8/19/2015 7:10:01 PM 61.6 ng/ul 1.233 0.686 1.80 1.02 DNA 50.00


Digestion of “pSB1C3 – mLacI miR373-BS” (19.08.2015)

Digestion
mLacI miR373-BS EcoRI-HF (NEB) XhoI (NEB) Cut Smart Buffer ddH₂O Total
1 3.4 ul 0.5 ul 0.5 ul 2.0 ul 13.9 ul 20.0 ul

37˚C 2.30h


Ligation of “pSB1C3 – mLacI miR373-BS with pTRE”

Ligation
Digested mLacI miR373-BS with pTRE pTRE T4 DNA Ligase Buffer T4 DNA Ligase Cut Smart Buffer ddH₂O Total
1 3.0 ul 1.0 ul 2.0 ul 1.0 ul 2.0 ul 13.0 ul 20.0 ul

22˚C 1.30h

Transformation will be made.


Colony PCR of “#6,8” (20.08.2015)

Colony PCR
MgCl₂ (NH₄)2SO₄ ColA fw ColA rev dNTP Tag ddH₂O DNA Total
1x 2.5 ul 2.5 ul 1.0 ul 1.0 ul 0.5 ul 0.2 ul 12.3 ul 5.0 ul 25.0 ul
25X 62.5 ul 62.5 ul 25.0 ul 25.0 ul 12.5 ul 5.0 ul 307.5 ul 500.0 ul
Cycling
95˚C 95˚C 56˚C 72˚C 72˚C Cycle
Time 5’ 30’’ 30’’ 1.5’ 5’ 35x

Result: Positive. Liquid Culture was made.


# Sample ID User name Date and Time Nucleic Acid Conc. Unit A260 A280 260/280 260/230 Sample Type Factor
1 blank biospec 8/22/2015 12:11:34 PM 0.5 ng/ul 0.010 -0.003 -3.39 -3.90 DNA 50.00
2 8-1 biospec 8/22/2015 12:12:38 PM 70.3 ng/ul 1.406 0.755 1.86 1.64 DNA 50.00
3 8-3 biospec 8/22/2015 12:13:38 PM 80.7 ng/ul 1.613 0.846 1.91 2.11 DNA 50.00
4 6-1 biospec 8/22/2015 12:14:32 PM 33.3 ng/ul 0.666 0.354 1.88 1.44 DNA 50.00


Cut Check of “#6,8”

Cut Check
DNA (400 ng/ul) EcoRI PstI Fast Digest Buffer ddH₂O Total
1 5.7 ul 0.5 ul 0.5 ul 2.0 ul 11.3 ul 20.0 ul
2 4.8 ul 0.5 ul 0.5 ul 2.0 ul 12.2 ul 20.0 ul
3 12.0 ul 0.5 ul 0.5 ul 2.0 ul 5.0 ul 20.0 ul

37˚C 40’

Result: positive

GEL GÖRÜNTÜSÜ


Colony PCR “pSB1C3 17,6,8 – pColA, Damp-Pex, pTetoff” (19.08.2015)

GEL GÖRÜNTÜLERI


Mini Prep of “pSB1C3-#17”

# Sample ID User name Date and Time Nucleic Acid Conc. Unit A260 A280 260/280 260/230 Sample Type Factor
1 blank biospec 8/20/2015 5:04:31 PM 0.6 ng/ul 0.013 0.007 1.93 1.40 DNA 50.00
2 psb1c3 17-2 biospec 8/20/2015 5:05:28 PM 78.9 ng/ul 1.579 0.821 1.92 2.17 DNA 50.00
3 psb1c3 17-5 biospec 8/20/2015 5:06:18 PM 77.5 ng/ul 1.551 0.806 1.92 2.08 DNA 50.00
4 psb1c3 17-8 biospec 8/20/2015 5:07:02 PM 87.5 ng/ul 1.749 0.910 1.92 2.08 DNA 50.00
5 psb1c3 17-10 biospec 8/20/2015 5:07:47 PM 105.4 ng/ul 2.108 1.087 1.94 2.15 DNA 50.00


Digestion of “pSB1C3 T7-10 and pET45 #4-1, #5-3, #6-6, #8-15” (21.08.2015)

Digestion pET45 #4-1, #5-3, #6-6, #8-15
DNA BamHI-HF (NEB) XhoI (NEB) Cut Smart Buffer ddH₂O Total
#4-1 10.1 ul 1.0 ul 1.0 ul 2.0 ul 5.9 ul 20.0 ul
#5-3 10.8 ul 1.0 ul 1.0 ul 2.0 ul 5.2 ul 20.0 ul
#6-6 5.0 ul 1.0 ul 1.0 ul 2.0 ul 11.0 ul 20.0 ul
#8-15 16.0 ul 1.0 ul 1.0 ul 2.0 ul - 20.0 ul

37˚C 3h


Digestion pSB1C3 T7-10
DNA BamHI-HF (NEB) XhoI (NEB) Cut Smart Buffer ddH₂O Total
#4-1 4.8 ul 1.0 ul 1.0 ul 2.0 ul 11.2 ul 20.0 ul

1ul rSAP / 37˚C 1h


Ligation
Vector (pSB1C3) Insert T4 DNA Ligase Buffer T4 DNA Ligase ddH₂O Total
#4 1.0 ul 1.0 ul 2.0 ul 1.0 ul 15.0 ul 20.0 ul
#5 1.0 ul 1.0 ul 2.0 ul 1.0 ul 15.0 ul 20.0 ul
#6 1.0 ul 1.0 ul 2.0 ul 1.0 ul 15.0 ul 20.0 ul
#8 1.0 ul 1.0 ul 2.0 ul 1.0 ul 15.0 ul 20.0 ul

22˚C 2h

-80 ˚C 20’’ Heat Inaktivation


Colony PCR of “pTRE - #13 mLacI-miR373 BS” (21.08.2015)

Colony PCR
MgCl₂ (NH₄)2SO₄ CMV fw SV40 rv dNTP Tag ddH₂O DNA Total
1x 2.5 ul 2.5 ul 1.0 ul 1.0 ul 0.5 ul 0.2 ul 12.3 ul 5.0 ul 25.0 ul
22X 55.0 ul 55.0 ul 22.0 ul 22.0 ul 11.0 ul 4.4 ul 270.6 ul 440.0 ul
Cycling
95˚C 95˚C 56˚C 72˚C 72˚C Cycle
Time 5’ 30’’ 30’’ 1.5’ 5’ 35x

Results: negative

GEL GÖRÜNTÜLERI


Mini Prep of “pSB1C3 #6-25,26”

# Sample ID User name Date and Time Nucleic Acid Conc. Unit A260 A280 260/280 260/230 Sample Type Factor
1 blank biospec 8/20/2015 10:01:33 AM 0.4 ng/ul 0.007 -0.003 -2.12 -1.13 DNA 50.00
2 psb1c3 #6-25 biospec 8/20/2015 10:02:45 AM 41.0 ng/ul 0.820 0.421 1.95 1.93 DNA 50.00
3 psb1c3 #6-26 biospec 8/20/2015 10:03:50 AM 46.0 ng/ul 0.920 0.464 1.98 1.97 DNA 50.00


Cut Check of “pSB1C3 #6-25,26”

Cut Check
DNA EcoRI-HF PstI-HF Cut Smart Buffer ddH₂O Total
#6-25 6.1 ul 0.5 ul 0.5 ul 2.0 ul 10.9 ul 20.0 ul
#6-26 5.5 ul 0.5 ul 0.5 ul 2.0 ul 11.5 ul 20.0 ul

37˚C 3h

Result: negative


Digestion of “pTRE #12”

Digestion
pTRE #12 (5000 ng/ul) EcoRI-HF XhoI (NEB) Cut Smart Buffer ddH₂O Total
1x 16.0 ul 0.5 ul 0.5 ul 2.0 ul 1.0 ul 20.0 ul

37˚C 3h

Result: negative

GEL GÖRÜNTÜSÜ


Digestion of “Damp-Pex pSB1C3” (22.08.2015)

Digestion pSB1C3 T7-10
DNA (500 ng/ul) NotI Cut Smart Buffer ddH₂O Total
#9-5 3.42 ul 1.0 ul 2.0 ul 13.58 ul 20.0 ul
#9-10 3.56 ul 1.0 ul 2.0 ul 13.44 ul 20.0 ul

37˚C 3h


Ligation of “Damp-Pex pColA”

Ligation
pColA (100 ng/ul) Insert (36 ng/ul) T4 DNA Ligase Buffer T4 DNA Ligase ddH₂O Total
1x 2.8 ul 5.41 ul 2.0 ul 1.0 ul 15.0 ul 20.0 ul

22˚C 1.30h

Transformation with NEB5α.


Colony PCR of “Damp-Pex + pColA” (23.08.2015)

Colony PCR
MgCl₂ (NH₄)2SO₄ pColA fw pColA rv dNTP Tag ddH₂O DNA Total
1x 2.5 ul 2.5 ul 1.0 ul 1.0 ul 0.5 ul 0.2 ul 12.3 ul 5.0 ul 25.0 ul
26X 65.0 ul 65.0 ul 26.0 ul 26.0 ul 13.0 ul 5.2 ul 319.8 ul 520.0 ul
Cycling
95˚C 95˚C 56˚C 72˚C 72˚C Cycle
Time 5’ 30’’ 30’’ 1.5’ 5’ 35x

Result: #5-4, #5-9 Liquid Culture was made.

GEL GÖRÜNTÜSÜ


Mini Prep of “pTetoff #14-4,5,8” (22.08.2015)

# Sample ID User name Date and Time Nucleic Acid Conc. Unit A260 A280 260/280 260/230 Sample Type Factor
1 blank biospec 8/22/2015 11:01:09 AM 0.1 ng/ul 0.002 -0.008 -0.23 -0.10 DNA 50.00
2 ptetoff 14-4 biospec 8/22/2015 11:02:05 AM 159.6 ng/ul 3.193 1.678 1.90 2.00 DNA 50.00
3 ptetoff 14-5 biospec 8/22/2015 11:03:21 AM 141.1 ng/ul 2.822 1.466 1.93 1.97 DNA 50.00
4 ptetoff 14-8 biospec 8/22/2015 11:04:14 AM 367.5 ng/ul 7.350 3.969 1.85 2.11 DNA 50.00


Cut Check I
DNA (250 ng/ul) SalI (FD) HindIII (FD) Fast Digest Buffer ddH₂O Total
#14-4 1.6 ul 0.5 ul 0.5 ul 2.0 ul 15.4 ul 20.0 ul
#14-5 1.8 ul 0.5 ul 0.5 ul 2.0 ul 15.2 ul 20.0 ul
#14-8 0.7 ul 0.5 ul 0.5 ul 2.0 ul 16.3 ul 20.0 ul

37˚C 2h

Result: negative

GEL GÖRÜNTÜSÜ


Cut Check II
DNA (250 ng/ul) SalI (FD) NotI (FD) Fast Digest Buffer ddH₂O Total
#14-4 1.6 ul 0.5 ul 0.5 ul 2.0 ul 15.4 ul 20.0 ul
#14-5 1.8 ul 0.5 ul 0.5 ul 2.0 ul 15.2 ul 20.0 ul
#14-8 0.7 ul 0.5 ul 0.5 ul 2.0 ul 16.3 ul 20.0 ul

37˚C 30’’

Result: negative

GEL GÖRÜNTÜSÜ


Colony PCR of “pSB1C3 – T7 #4,5,6,8” (23.08.2015)

Colony PCR
MgCl₂ (NH₄)2SO₄ pTRE Luc fw SV40 rv dNTP Tag ddH₂O DNA Total
1x 2.5 ul 2.5 ul 1.0 ul 1.0 ul 0.5 ul 0.2 ul 12.3 ul 5.0 ul 25.0 ul
50x 125.0 ul 125.0 ul 50.0 ul 50.0 ul 25.0 ul 10.0 ul 615.0 ul 1000.0 ul
Cycling
95˚C 95˚C 56˚C 72˚C 72˚C Cycle
Time 5’ 30’’ 30’’ 1.5’ 5’ 35x

GEL GÖRÜNTÜSÜ </div> </section> 

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           <label for="august-week4">Week 1, 24.08.-31.08.</label>
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</html> Digestion of “pTRE #12-2” (24.08.2015)

Digestion
pTRE #12-2 (2000 ng/ul) EcoRI-HF XhoI-NEB Cut Smart Buffer ddH₂O Total
1x 6.4 ul 0.5 ul 0.5 ul 2.0 ul 10.6 ul 20.0 ul

37˚C 2h

Result: Bands were at the expected section. Gel Extraction was made.


Mini Prep

# Sample ID User name Date and Time Nucleic Acid Conc. Unit A260 A280 260/280 260/230 Sample Type Factor
1 blank biospec 8/24/2015 1:28:49 AM 0.0 ng/ul 0.001 -0.013 -0.07 -0.07 DNA 50.00
2 ptre gel biospec 8/24/2015 1:30:29 AM 15.3 ng/ul 0.306 0.164 1.87 0.65 DNA 50.00


# Sample ID User name Date and Time Nucleic Acid Conc. Unit A260 A280 260/280 260/230 Sample Type Factor
1 blank biospec 8/24/2015 1:26:27 AM 0.7 ng/ul 0.015 0.001 18.47 0.53 DNA 50.00
2 ptre biospec 8/24/2015 1:29:38 AM 13.7 ng/ul 0.275 0.145 1.90 0.10 DNA 50.00


Digestion of “TEV-Protease pET45/pSB1C3 T7/pColA (24.08.2015)

Digestion
DNA (500 ng/ul) BamHI-HF XhoI-HF Cut Smart Buffer ddH₂O Total
1x 5.86 ul 0.5 ul 0.5 ul 2.0 ul 11.14 ul 20.0 ul

37˚C 3h


Ligation of “pCAGop with pTRE #12-2” (24.08.2015)

Ligation
pCAG (18.38 ng/ul) pTRE #12-2 (100 ng/ul) T4 DNA Ligase T4 DNA Ligase Buffer Cut Smart Buffer ddH₂O Total
1x 0.6 ul 6.6 ul 1.0 ul 2.0 ul 2.0 ul 9.8 ul 20.0 ul

Transformation was made.


Cut Check “pSB1C3-T7 #4,5,6,8” (24.08.2015)

Cut Check
DNA (300 ng/ul) EcoRI (FD) PstI (FD) Fast Digest Buffer ddH₂O Total
1x 3.5 ul 1.0 ul 1.0 ul 2.0 ul 12.5 ul 20.0 ul

30’ digestion


# Sample ID User name Date and Time Nucleic Acid Conc. Unit A260 A280 260/280 260/230 Sample Type Factor
1 blank biospec 8/25/2015 1:26:12 PM 0.8 ng/ul 0.016 -0.003 -5.85 2.25 DNA 50.00
2 #9-5-4 biospec 8/25/2015 1:27:29 PM 59.1 ng/ul 1.182 0.603 1.96 2.25 DNA 50.00
3 #9-5-9 biospec 8/25/2015 1:28:30 PM 37.9 ng/ul 0.758 0.375 2.02 2.19 DNA 50.00


Colony PCR of “pTRE #13” (25.08.2015)

Colony PCR
MgCl₂ (NH₄)2SO₄ CMV fw SV40 rv dNTP Tag ddH₂O DNA Total
1x 2.5 ul 2.5 ul 1.0 ul 1.0 ul 0.5 ul 0.2 ul 12.3 ul 5.0 ul 25.0 ul
7x 17.5 ul 17.5 ul 7.0 ul 7.0 ul 3.5 ul 1.4 ul 86.1 ul 140.0 ul
Cycling
95˚C 95˚C 56˚C 72˚C 72˚C Cycle
Time 5’ 30’’ 30’’ 1.5’ 5’ 35x

GEL GÖRÜNTÜSÜ + pTRE13 cut check görüntüsü


Colony PCR of “pCAG-DsRed” (25.08.2015)

Colony PCR
MgCl₂ (NH₄)2SO₄ pTRE Luc fw SV40 rv dNTP Tag ddH₂O DNA Total
1x 2.5 ul 2.5 ul 1.0 ul 1.0 ul 0.5 ul 0.2 ul 12.3 ul 5.0 ul 25.0 ul
14x 35.0 ul 35.0 ul 14.0 ul 14.0 ul 7.0 ul 2.8 ul 172.2 ul 280.0 ul
Cycling
95˚C 95˚C 56˚C 72˚C 72˚C Cycle
Time 5’ 30’’ 30’’ 1.5’ 5’ 35x

Result: Bands weren’t at the expected section.

GEL GÖRÜNTÜSÜ


Cut Check of „pSB1C3 – TlpB“ (25.05.2015)

Cut Check
DNA (250 ng/ul) EcoRI (FD) Fast Digest Buffer ddH₂O Total
#8-1 3.6 ul 0.5 ul 2.0 ul 13.9 ul 20.0 ul
#8-3 3.1 ul 0.5 ul 2.0 ul 14.4 ul 20.0 ul

7˚C 35’

The colonies will be made glycerol stock.

GEL GÖRÜNTÜSÜ


Ligation of “Damp-Pex” (25.08.2015)

Ligation
DNA (500 ng/ul) NotI Cut Smart Buffer ddH₂O Total
#9-5 3.78 ul 1.0 ul 2.0 ul 13.22 ul 20.0 ul
#9-10 3.87 ul 1.0 ul 2.0 ul 13.13 ul 20.0 ul

37˚C 3h


Ligation
ColA (100 ng/ul) Insert (36 ng/ul) T4 DNA Ligase T4 DNA Ligase Buffer ddH₂O Total
#9-5 10.0 ul 14.4 ul 1.0 ul 3.0 ul 1.6 ul 30.0 ul

22˚C 1.5h

Transformation was made.


Colony PCR of “pCAGop-DsRed” (26.08.2015)

Colony PCR
MgCl₂ (NH₄)2SO₄ pTRE Luc fw SV40 rv dNTP Tag ddH₂O DNA Total
1x 2.5 ul 2.5 ul 1.0 ul 1.0 ul 0.5 ul 0.2 ul 12.3 ul 5.0 ul 25.0 ul
10x 25.0 ul 25.0 ul 10.0 ul 10.0 ul 5.0 ul 2.0 ul 123.0 ul 200.0 ul
Cycling
95˚C 95˚C 56˚C 72˚C 72˚C Cycle
Time 5’ 30’’ 30’’ 1.5’ 5’ 35x

Result: 2 colonie will be made liquid culture and streak plate. Ligation and transformation will be repeated.

GEL GÖRÜNTÜSÜ


Mini Prep of “pTRE #13-3,5” (26.08.2015)

# Sample ID User name Date and Time Nucleic Acid Conc. Unit A260 A280 260/280 260/230 Sample Type Factor
1 blank biospec 8/26/2015 4:55:28 PM 0.1 ng/ul 0.003 -0.020 -0.13 -0.13 DNA 50.00
2 ptre 13-3 biospec 8/26/2015 4:56:47 PM 77.9 ng/ul 1.558 0.772 2.02 1.67 DNA 50.00
3 ptre 13-5 biospec 8/26/2015 4:58:06 PM 76.0 ng/ul 1.520 0.776 1.96 1.48 DNA 50.00


Colony PCR of “pColA – Damp-Pex, pSB1C3 – TEV

Colony PCR “pColA – Damp-Pex”
MgCl₂ (NH₄)2SO₄ pTRE Luc fw SV40 rv dNTP Tag ddH₂O DNA Total
1x 2.5 ul 2.5 ul 1.0 ul 1.0 ul 0.5 ul 0.2 ul 12.3 ul 5.0 ul 25.0 ul
10x 25.0 ul 25.0 ul 10.0 ul 10.0 ul 5.0 ul 2.0 ul 123.0 ul 200.0 ul
Cycling
95˚C 95˚C 56˚C 72˚C 72˚C Cycle
Time 5’ 30’’ 30’’ 1.5’ 5’ 35x


Colony PCR “pSB1C3 – TEV Protease”

Colony PCR “pSB1C3 – TEV Protease”
MgCl₂ (NH₄)2SO₄ VR VF dNTP Tag ddH₂O DNA Total
1x 2.5 ul 2.5 ul 1.0 ul 1.0 ul 0.5 ul 0.2 ul 12.3 ul 5.0 ul 25.0 ul
10x 25.0 ul 25.0 ul 10.0 ul 10.0 ul 5.0 ul 2.0 ul 123.0 ul 200.0 ul
Cycling
95˚C 95˚C 56˚C 72˚C 72˚C Cycle
Time 5’ 30’’ 30’’ 1.5’ 5’ 35x

Result: negative

GEL GÖRÜNTÜSÜ


Colony PCR “pSB1C3 – TEV Protease” – Repeat (27.08.2015)

Colony PCR “pSB1C3 – TEV Protease”
MgCl₂ (NH₄)2SO₄ VR VF dNTP Tag ddH₂O DNA Total
1x 2.5 ul 2.5 ul 1.0 ul 1.0 ul 0.5 ul 0.2 ul 12.3 ul 5.0 ul 25.0 ul
15x 37.5 ul 37.5 ul 15.0 ul 15.0 ul 7.5 ul 3.0 ul 184.5 ul 300.0 ul
Cycling
95˚C 95˚C 56˚C 72˚C 72˚C Cycle
Time 5’ 30’’ 30’’ 1.5’ 5’ 35x

Result: negative

GEL GÖRÜNTÜSÜ


Colony PCR of “pTRE – Luc #14” (27.08.2015)

Colony PCR
MgCl₂ (NH₄)2SO₄ pTRE Luc fw pTRE Luc rv dNTP Tag ddH₂O DNA Total
1x 2.5 ul 2.5 ul 1.0 ul 1.0 ul 0.5 ul 0.2 ul 12.3 ul 5.0 ul 25.0 ul
14x 35.0 ul 35.0 ul 14.0 ul 14.0 ul 7.0 ul 2.8 ul 172.2 ul 280.0 ul
Cycling
95˚C 95˚C 56˚C 72˚C 72˚C Cycle
Time 5’ 30’’ 30’’ 1.5’ 5’ 35x

Result: Bands weren’t at the expected section.

GEL GÖRÜNTÜSÜ


Gel Extraction of “Damp-Pex”

# Sample ID User name Date and Time Nucleic Acid Conc. Unit A260 A280 260/280 260/230 Sample Type Factor
1 blank biospec 8/27/2015 9:39:17 PM 1.1 ng/ul 0.022 0.009 2.36 1.00 DNA 50.00
2 damp-pex biospec 8/27/2015 9:40:04 PM 8.2 ng/ul 0.164 0.090 1.82 0.05 DNA 50.00


Colony PCR of “pCAGop-DsRed #12” (27.08.2015)

Colony PCR
MgCl₂ (NH₄)2SO₄ pTRE Luc fw SV40 rv dNTP Tag ddH₂O DNA Total
1x 2.5 ul 2.5 ul 1.0 ul 1.0 ul 0.5 ul 0.2 ul 12.3 ul 5.0 ul 25.0 ul
14x 35.0 ul 35.0 ul 14.0 ul 14.0 ul 7.0 ul 2.8 ul 172.2 ul 280.0 ul
Cycling
95˚C 95˚C 56˚C 72˚C 72˚C Cycle
Time 5’ 30’’ 30’’ 1.5’ 5’ 35x

Result: Negative.

GEL GÖRÜNTÜSÜ


Ligation of “pCAGop-DsRed” (27.08.2015)

Ligation
pCAG (18.38 ng/ul) pTRE #12 (100 ng/ul) T4 DNA Ligase T4 DNA Ligase Buffer ddH₂O Total
1x 0.3 ul 6.6 ul 1.0 ul 2.0 ul 10.1 ul 20.0 ul

22˚C 1.30h

Transformation was made.


Colony PCR of „pSB1C3-T7 #5“ (27.08.2015)

Colony PCR
MgCl₂ (NH₄)2SO₄ pTRE Luc fw SV40 rv dNTP Tag ddH₂O DNA Total
1x 2.5 ul 2.5 ul 1.0 ul 1.0 ul 0.5 ul 0.2 ul 12.3 ul 5.0 ul 25.0 ul
8x 20.0 ul 20.0 ul 8.0 ul 8.0 ul 4.0 ul 1.6 ul 98.4 ul 160.0 ul
Cycling
95˚C 95˚C 56˚C 72˚C 72˚C Cycle
Time 5’ 30’’ 30’’ 1.5’ 5’ 35x

Result: #1-1 Liquid Culture was made.

GEL GÖRÜNTÜSÜ


Digestion of “pSB1C3-T7 TEV Protease (17. and 18. Colony)” (27.08.2015)

Digestion
DNA (500 ng/ul) NotI-HF Cut Smart Buffer ddH₂O Total
17 6.0 ul 0.5 ul 2.0 ul 11.5 ul 20.0 ul
18 13.0 ul 0.5 ul 2.0 ul 4.5 ul 20.0 ul

37˚C 3h

65˚C 20’’ Heat Inactivation

Result: negative

GEL GÖRÜNTÜSÜ


Digestion of “pColA” (27.08.2015)

Digestion
pColA (300 ng/ul) NotI Buffer ddH₂O Total
1x 11.76 ul 0.5 ul 2.0 ul 5.74 ul 20.0 ul

37˚C 3h

1.2 ul rSAP

37˚C 1h

20’’ Heat Inactivation


Colony PCR of “pCAGop-DsRed #12-2. Colony streak” (28.08.2015)

Colony PCR
MgCl₂ (NH₄)2SO₄ pTRE Luc fw SV40 rv dNTP Tag ddH₂O DNA Total
1x 2.5 ul 2.5 ul 1.0 ul 1.0 ul 0.5 ul 0.2 ul 12.3 ul 5.0 ul 25.0 ul
12x 30.0 ul 30.0 ul 12.0 ul 12.0 ul 6.0 ul 2.4 ul 147.6 ul 240.0 ul
Cycling
95˚C 95˚C 56˚C 72˚C 72˚C Cycle
Time 5’ 30’’ 30’’ 1.5’ 5’ 35x

Result: Negative

GEL GÖRÜNTÜSÜ


Colony PCR of „pSB1C3-T7 #5“ (28.08.2015)

Colony PCR
MgCl₂ (NH₄)2SO₄ VF VR dNTP Tag ddH₂O DNA Total
1x 2.5 ul 2.5 ul 1.0 ul 1.0 ul 0.5 ul 0.2 ul 12.3 ul 5.0 ul 25.0 ul
16x 40.0 ul 40.0 ul 16.0 ul 16.0 ul 13.0 ul 3.2 ul 196.8 ul 325.0 ul
Cycling
95˚C 95˚C 56˚C 72˚C 72˚C Cycle
Time 5’ 30’’ 30’’ 1.5’ 5’ 35x

Result: #5-31 Bands were at the expected section

GEL GÖRÜNTÜSÜ


Cut Check of „pSB1C3-T7 #5-31“

Cut Check
DNA EcoRI (FD) PstI (FD) Fast Digest Buffer ddH₂O Total
1x ??????? 1.0 ul 1.0 ul 2.0 ul ????? 20.0 ul

GEL GÖRÜNTÜSÜ

Digestion of “pSB1C3 #9” (28.08.2015)

Digestion
DNA (500 ng/ul) NotI Cut Smart Buffer ddH₂O Total
1x ??????? 1.0 ul 2.0 ul ????? 20.0 ul

3h was digested

Result: #9-5 Bands were at the expected section

GEL GÖRÜNTÜSÜ


Ligation of “pColA #9”

Ligation
pColA #9 T4 DNA Ligase T4 DNA Ligase Buffer ddH₂O Total
1x 7.5 ul 9.2 ul 1.0 ul 2.0 ul 0.3 ul 20.0 ul

2h was ligasted


Mini Prep of “pCAG-DsRed #12” (28.08.2015)

# Sample ID User name Date and Time Nucleic Acid Conc. Unit A260 A280 260/280 260/230 Sample Type Factor
1 blank biospec 8/28/2015 7:23:31 PM 0.5 ng/ul 0.010 -0.006 -1.58 -1.46 DNA 50.00
2 pcag op-dsred biospec 8/28/2015 7:24:38 PM 92.3 ng/ul 1.846 1.105 1.67 0.79 DNA 50.00
3 pcag op-dsred biospec 8/28/2015 7:26:20 PM 67.4 ng/ul 1.348 0.702 1.92 1.35 DNA 50.00


Cut Check “pCAGop-DsRed #12”

Cut Check
DNA EcoRI-HF XhoI (NEB) Cut Smart Buffer ddH₂O Total
1x 3.7 ul 0.5 ul 0.5 ul 2.0 ul 13.3 ul 20.0 ul

Result: negative

GEL GÖRÜNTÜSÜ


Cut Check “pCAGop-DsRed #12” - Repeat

Cut Check
DNA EcoRI-HF XhoI (NEB) Cut Smart Buffer ddH₂O Total
1x 3.7 ul 0.5 ul 0.5 ul 2.0 ul 13.3 ul 20.0 ul

Result: negative


Colony PCR of „pTEToff and pTEToff N term“ (28.08.2015)

Colony PCR
MgCl₂ (NH₄)2SO₄ CMV rv pTRE Luc fw dNTP Tag ddH₂O DNA Total
1x 2.5 ul 2.5 ul 1.0 ul 1.0 ul 0.5 ul 0.2 ul 12.3 ul 5.0 ul 25.0 ul
26x 65.0 ul 65.0 ul 26.0 ul 26.0 ul 13.0 ul 5.2 ul 319.8 ul 520.0 ul
Cycling
95˚C 95˚C 56˚C 72˚C 72˚C Cycle
Time 5’ 30’’ 30’’ 1.5’ 5’ 35x

Result: positive

GEL GÖRÜNTÜSÜ


Mini Prep of “pSB1C3-T7 TEV Protease” (28.09.2015)

# Sample ID User name Date and Time Nucleic Acid Conc. Unit A260 A280 260/280 260/230 Sample Type Factor
1 blank biospec 8/28/2015 5:55:09 PM 0.0 ng/ul -0.001 -0.010 0.10 -1.29 DNA 50.00
2 tev psb1c3-t7/17 biospec 8/28/2015 5:56:45 PM 81.8 ng/ul 1.637 0.848 1.93 2.08 DNA 50.00
3 tev psb1c3-t7/18 biospec 8/28/2015 5:59:01 PM 38.5 ng/ul 0.771 0.380 2.03 2.03 DNA 50.00


Mini Prep of “pSB1C3-T7 TEV Protease” (29.09.2015)

# Sample ID User name Date and Time Nucleic Acid Conc. Unit A260 A280 260/280 260/230 Sample Type Factor
1 blank biospec 8/29/2015 8:08:11 PM -0.8 ng/ul -0.016 -0.019 0.83 0.68 DNA 50.00
2 psb1c3-t7/tev17 biospec 8/29/2015 8:09:26 PM 135.4 ng/ul 2.709 1.355 2.00 1.68 DNA 50.00
3 psb1c3-t7/tev18 biospec 8/29/2015 8:11:06 PM 229.1 ng/ul 4.581 2.193 2.09 2.40 DNA 50.00


Mini Prep of “pCAGop-DsRed #1-1” (30.08.2015)

# Sample ID User name Date and Time Nucleic Acid Conc. Unit A260 A280 260/280 260/230 Sample Type Factor
1 blank biospec 8/30/2015 11:05:33 AM 0.2 ng/ul 0.003 -0.012 -0.25 -0.27 DNA 50.00
2 pcag op-dsred biospec 8/30/2015 11:07:31 AM 79.8 ng/ul 1.595 0.823 1.94 1.72 DNA 50.00


Cut Check of “pCAGop-DsRed #1-1”

Cut Check
pCAGop-DsRed (250 ng/ul) EcoRI-HF XhoI (NEB) Cut Smart Buffer ddH₂O Total
1x 3.2 ul 0.5 ul 0.5 ul 2.0 ul 13.8 ul 20.0 ul

37˚C 3h

Result: negative

GEL GÖRÜNTÜSÜ


Digestion of “pSB1C3-T7 TEV Protease” (30.08.2015)

Digestion
Insert NotI Cut Smart Buffer ddH₂O Total
17 3.7 ul 0.25 ul 2.0 ul 14.05 ul 20.0 ul
18 2.2 ul 0.25 ul 2.0 ul 15.55 ul 20.0 ul

37˚C 3h

Result: Bands were at the expected section. Gel extraction was made.

GEL GÖRÜNTÜSÜ


Ligation of “pSB1C3-T7 TEV Protease”

Ligation
Vector (60 ng/ul) Insert (29.11 ng/ul) T4 DNA Buffer T4 DNA Buffer Ligase ddH₂O Total
1x 7.0 ul 8.7 ul 2.0 ul 1.0 ul 1.3 ul 20.0 ul

22˚C 1.5h

Transformation was made.


Gel Extraction

# Sample ID User name Date and Time Nucleic Acid Conc. Unit A260 A280 260/280 260/230 Sample Type Factor
1 blank biospec 8/30/2015 5:28:51 PM 0.0 ng/ul -0.001 -0.005 0.15 0.03 DNA 50.00
2 psb1c3-t7 biospec 8/30/2015 5:28:05 PM 5.6 ng/ul 0.113 0.061 1.85 0.17 DNA 50.00


Colony PCR of “pColA – TEV” (31.08.2015)

Colony PCR
MgCl₂ (NH₄)2SO₄ pColA fw pColA rv dNTP Tag ddH₂O DNA Total
1x 2.5 ul 2.5 ul 1.0 ul 1.0 ul 0.5 ul 0.2 ul 12.3 ul 5.0 ul 25.0 ul
16x 40.0 ul 40.0 ul 16.0 ul 16.0 ul 8.0 ul 3.2 ul 196.8 ul 320.0 ul
Cycling
95˚C 95˚C 56˚C 72˚C 72˚C Cycle
Time 5’ 30’’ 30’’ 1.5’ 5’ 35x

Result: negative

GEL GÖRÜNTÜSÜ


Colony PCR of “pColA – TEV” – Repeat (31.08.2015)

Colony PCR
MgCl₂ (NH₄)2SO₄ pColA fw pColA rv dNTP Tag ddH₂O DNA Total
1x 2.5 ul 2.5 ul 1.0 ul 1.0 ul 0.5 ul 0.2 ul 12.3 ul 5.0 ul 25.0 ul
22x 55.0 ul 55.0 ul 22.0 ul 22.0 ul 11.0 ul 3.2 ul 270.6 ul 440.0 ul
Cycling
95˚C 95˚C 56˚C 72˚C 72˚C Cycle
Time 5’ 30’’ 30’’ 1.5’ 5’ 35x

Result: negative

GEL GÖRÜNTÜSÜ


Digestion of “PsicA for pSB1C3 and invF-sicA for ColA” (31.08.2015)

Digestion of “PsicA and invF”
DNA EcoRI-HF PstI XbaI SpeI 2.1 Buffer Cut Smart Buffer ddH₂O Total
invF-sicA 10.0 ul - - 1.0 ul 1.0 ul - 2.0 ul 6.0 ul 20.0 ul
PsicA 10.0 ul 1.0 ul 1.0 ul - - 2.0 ul - 6.0 ul 20.0 ul

37˚C 10h

82˚C 20’ Heat Inactivation


Digestion of “pSB1C3-RFP and pColA-NotI”
DNA EcoRI (FD) PstI (FD) XbaI (FD) SpeI (FD) Fast Digest Buffer ddH₂O Total
pSB1C3-RFP (1000 ng/ul) 2.5 ul 1.0 ul 1.0 ul - - 2.0 ul 13.5 ul 20.0 ul
pColA-NotI (500 ng/ul) 5.0 ul - - 1.0 ul 1.0 ul 2.0 ul 11.0 ul 20.0 ul

September

02.09.2015 Wiki meetings continue, we are making decisions together. 06.09.2015 What will we be wearing at the Giant Jamboree? Thinking about how to design our T-shirts. 08.09.2015 Team pictures mustn’t be left ends. 11.09.2015 We taught our little friends synthetic biology. Those kids were really funny when they came first, they were covering their faces because they were scared of being contaminated. 13.09.2015 Coloring our drawings is complete. They are better than original now. We still have wiki meetings, there are only 5 days to wiki-freeze!

Ligation of “ColA and pSB1C3” (01.09.2015)

Ligation
Vector (100 ng/ul) invF-sicA psicA T4 DNA Ligase T4 DNA Ligase Buffer ddH₂O Total
ColA 8.5 ul 17.5 ul - 1.0 ul 2.0 ul - 30.0 ul
pSB1C3 9.44 ul - 13.55 ul 1.0 ul 2.0 ul 3.0 ul 30.0 ul

22˚C 2h


# Sample ID User name Date and Time Nucleic Acid Conc. Unit A260 A280 260/280 260/230 Sample Type Factor
1 blank biospec 9/1/2015 4:10:17 AM 0.3 ng/ul 0.006 -0.007 -0.88 -0.78 DNA 50.00
2 colA ext biospec 9/1/2015 4:11:28 AM 11.7 ng/ul 0.235 0.132 1.78 0.08 DNA 50.00
3 cola clean-up biospec 9/1/2015 4:12:56 AM 7.2 ng/ul 0.143 0.078 1.84 0.06 DNA 50.00
4 psb1c3 ext biospec 9/1/2015 4:14:40 AM 10.6 ng/ul 0.213 0.113 1.88 0.12 DNA 50.00

Transformation will be made.


Ligation of “pCAGop-DsRed” (01.09.2015)

Ligation
pCAG pTRE #12 T4 DNA Ligase T4 DNA Ligase Buffer ddH₂O Total
1x 0.3 ul 4.0 ul 1.0 ul 1.0 ul 13.7 ul 20.0 ul

Transformation with BL21.


Digestion of “pSB1C3-T7 TEV and pColA” (01.09.2015)

Digestion
DNA XhoI-HF SpeI-HF Cut Smart Buffer RNAse ddH₂O Total
17 (Hand Made Solution) 10.0 ul 0.25 ul 0.25 ul 2.0 ul 0.2 ul 7.3 ul 20.0 ul
18 (Hand Made Solution) 10.0 ul 0.25 ul 0.25 ul 2.0 ul 0.2 ul 7.3 ul 20.0 ul
17 (Midi Prep Solution) 10.0 ul 0.25 ul 0.25 ul 2.0 ul - 7.5 ul 20.0 ul
18 (Midi Prep Solution) 10.0 ul 0.25 ul 0.25 ul 2.0 ul - 7.5 ul 20.0 ul
pColA (1000 ng/ul) 9.0 ul 0.5 ul 0.5 ul 2.0 ul - 8.0 ul

37˚C 3h

80˚C 20’ Heat Inactivation

Cut Check was made.

Görüntü

Result: pColA Bands were at the expected section. Midi Prep Solution is better.


Colony PCR of “pCAGop-DsRed and pCMV-lacO” (02.09.2015)

Colony PCR
MgCl₂ (NH₄)2SO₄ pTRE Luc fw SV40 rv dNTP Tag ddH₂O DNA Total
1x 2.5 ul 2.5 ul 1.0 ul 1.0 ul 0.5 ul 0.2 ul 12.3 ul 5.0 ul 25.0 ul
16x 40.0 ul 40.0 ul 16.0 ul 16.0 ul 8.0 ul 3.2 ul 196.8 ul 320.0 ul
Cycling
95˚C 95˚C 56˚C 72˚C 72˚C Cycle
Time 5’ 30’’ 30’’ 1.5’ 5’ 35x

Result: negative. It will be repeated.

GEL GÖRÜNTÜSÜ


Colony PCR of “invF-sicA and PsicA” (02.09.2015)

Result: PsicA: 1, 2, 6, 8, 9, 10, 11 Liquid Culture will be made.

GEL GÖRÜNTÜSÜ


Digestion of “invF-sicA for pSB1C3 and ColA” (02.09.2015)

DNA 10.0 ul + rSAP 1.0 ul / 37˚C 1h / 80˚C 20’

Digestion
DNA EcoRI-HF PstI XbaI SpeI 2.1 Buffer Cut Smart Buffer ddH₂O Total
pSB1C3 10.0 ul 0.4 ul 0.4 ul - - 2.0 ul - 7.2 ul 20.0 ul
ColA 10.0 ul - - 0.4 ul 0.4 ul - 2.0 ul 7.2 ul 20.0 ul

37˚C overnight


Mini Prep of “PsicA – pSB1C3” (03.08.2015)

# Sample ID User name Date and Time Nucleic Acid Conc. Unit A260 A280 260/280 260/230 Sample Type Factor
1 blank biospec 9/3/2015 8:34:29 PM 0.8 ng/ul 0.016 0.000 116.17 0.85 DNA 50.00
2 psb1c3-psica 1 biospec 9/3/2015 8:35:32 PM 130.5 ng/ul 2.610 1.370 1.91 2.04 DNA 50.00
3 psb1c3-psica 2 biospec 9/3/2015 8:36:34 PM 140.9 ng/ul 2.818 1.457 1.93 2.16 DNA 50.00
4 psb1c3-psica 6 biospec 9/3/2015 8:37:28 PM 178.5 ng/ul 3.569 1.864 1.92 2.20 DNA 50.00
5 psb1c3-psica 8 biospec 9/3/2015 8:38:24 PM 98.7 ng/ul 1.974 1.021 1.93 2.13 DNA 50.00
6 psb1c3-psica 9 biospec 9/3/2015 8:39:21 PM 237.0 ng/ul 4.741 2.476 1.91 2.13 DNA 50.00
7 psb1c3-psica 10 biospec 9/3/2015 8:40:35 PM 109.0 ng/ul 2.181 1.127 1.94 2.05 DNA 50.00
8 biospec 9/3/2015 8:41:51 PM 109.5 ng/ul 2.189 1.134 1.93 1.97 DNA 50.00
9 blank biospec 9/3/2015 8:43:10 PM 1.3 ng/ul 0.025 0.003 9.26 0.73 DNA 50.00
10 psb1c3-psica 11 biospec 9/3/2015 8:44:14 PM 50.4 ng/ul 1.008 0.518 1.95 1.97 DNA 50.00


Cut Check of “PsicA – pSB1C3”

Cut Check
DNA EcoRI (FD) PstI (FD) Fast Digest Buffer ddH₂O Total
1 1.9 ul 0.35 ul 0.35 ul 2.0 ul 15.4 ul 20.0 ul
2 1.8 ul 0.35 ul 0.35 ul 2.0 ul 15.5 ul 20.0 ul
6 1.4 ul 0.35 ul 0.35 ul 2.0 ul 15.9 ul 20.0 ul
8 2.5 ul 0.35 ul 0.35 ul 2.0 ul 14.8 ul 20.0 ul
9 1.1 ul 0.35 ul 0.35 ul 2.0 ul 16.2 ul 20.0 ul
10 2.3 ul 0.35 ul 0.35 ul 2.0 ul 15.0 ul 20.0 ul
11 5.0 ul 0.35 ul 0.35 ul 2.0 ul 12.3 ul 20.0 ul

37˚C 35’


Ligation of “invF and pSB1C3/pColA” (03.08.2015)

Ligation
Vector (100 ng/ul) invF/sicA invF/sicA T4 DNA Ligase T4 DNA Ligase Buffer ddH₂O Total
ColA (-) 11.6 ul - - 1.0 ul 3.2 ul 16.6 ul 32.2 ul
ColA (+) 11.6 ul 14.40 ul - 3.2 ul 3.4 ul 2.2 ul 34.8 ul
pSB1C3 9.4 ul - 14.40 ul 1.0 ul 3.0 ul 2.2 ul 30.0 ul

RT 1h

Transformation will be made.


Colony PCR of “pCMV-Lacop-DsRed and pCAGop-DsRed” (04.09.2015)

Colony PCR
MgCl₂ (NH₄)2SO₄ pTRE Luc fw SV40 rv dNTP Tag ddH₂O DNA Total
1x 2.5 ul 2.5 ul 1.0 ul 1.0 ul 0.5 ul 0.2 ul 12.3 ul 5.0 ul 25.0 ul
15x 37.5 ul 37.5 ul 15.0 ul 15.0 ul 7.5 ul 3.0 ul 184.5 ul 300.0 ul
Cycling
95˚C 95˚C 56˚C 72˚C 72˚C Cycle
Time 5’ 30’’ 30’’ 1.5’ 5’ 35x

Result: Bands were at the expected section. Liquid Culture was made.

GEL GÖRÜNTÜSÜ


Mini Prep of „pCMV-Lacop-DsRed and pCAGop-DsRed“ (05.09.2015)

# Sample ID User name Date and Time Nucleic Acid Conc. Unit A260 A280 260/280 260/230 Sample Type Factor
1 blank biospec 9/5/2015 10:42:13 AM -0.1 ng/ul -0.003 -0.015 0.18 0.15 DNA 50.00
2 pcmv-lacop-dsred 1 biospec 9/5/2015 10:44:11 AM 68.4 ng/ul 1.369 0.690 1.98 1.76 DNA 50.00
3 pcmv-lacop-dsred 2 biospec 9/5/2015 10:45:12 AM 51.2 ng/ul 1.024 0.499 2.05 1.77 DNA 50.00
4 pcmv-lacop-dsred 3 biospec 9/5/2015 10:46:09 AM 78.5 ng/ul 1.570 0.793 1.98 1.98 DNA 50.00
5 pcmv-lacop-dsred 4 biospec 9/5/2015 10:47:04 AM 47.7 ng/ul 0.954 0.460 2.07 1.95 DNA 50.00
6 Pcagop-dsred 1 biospec 9/5/2015 10:48:08 AM 499.6 ng/ul 9.991 5.290 1.89 2.20 DNA 50.00


Cut Check of „pCMV-Lacop-DsRed and pCAGop-DsRed“

Cut Check
DNA BamHI XhoI EcoRI Cut Smart Buffer ddH₂O Total
1 4.4 ul 0.5 ul 0.5 ul - 2.0 ul 12.6 ul 20.0 ul
2 5.9 ul 0.5 ul 0.5 ul - 2.0 ul 11.1 ul 20.0 ul
3 3.9 ul 0.5 ul 0.5 ul - 2.0 ul 13.1 ul 20.0 ul
4 6.3 ul 0.5 ul 0.5 ul - 2.0 ul 10.7 ul 20.0 ul
pCAGop-DsRed 1.0 ul 0.5 ul - 0.5 ul 2.0 ul
16.0 ul 20.0 ul

37˚C 3h

Result: 1-2-3-4 is positive.

GEL GÖRÜNTÜSÜ


Cut Check of “invF and sicA” (07.09.2015)

Cut Check
DNA (250 ng/ul) EcoRI (FD) PstI (FD) Fast Digest Buffer ddH₂O Total
1x 5.0 ul 0.5 ul 0.5 ul 2.0 ul 12.0 ul 20.0 ul

37˚C 35’

GEL GÖRÜNTÜSÜ


Mini Prep

# Sample ID User name Date and Time Nucleic Acid Conc. Unit A260 A280 260/280 260/230 Sample Type Factor
1 blank biospec 9/7/2015 6:34:56 PM -0.1 ng/ul -0.003 -0.005 0.60 0.11 DNA 50.00
2 #18-11 biospec 9/7/2015 6:36:20 PM 79.8 ng/ul 1.597 0.843 1.89 1.93 DNA 50.00
3 #18-2 biospec 9/7/2015 6:37:48 PM 74.9 ng/ul 1.497 0.787 1.90 2.01 DNA 50.00
4 elution blank biospec 9/7/2015 6:39:44 PM 0.3 ng/ul 0.005 -0.004 -1.51 0.68 DNA 50.00
5 #18-5 elution biospec 9/7/2015 6:40:49 PM 57.7 ng/ul 1.154 0.604 1.91 1.91 DNA 50.00


Digestion of “#18-2,5”

Digestion
DNA (500 ng/ul) XbaI SpeI-HF Cut Smart Buffer ddH₂O Total
#18-2 6.66 ul 0.4 ul 0.4 ul 2.0 ul 8.54 ul 20.0 ul
#18-5 8.66 ul 0.4 ul 0.4 ul 2.0 ul 10.54 ul 20.0 u

37˚C 40’


Ligation

Ligation
pSB1C3-invF (175 ng/ul) ColA (100 ng/ul) T4 DNA Ligase T4 DNA Ligase Buffer ddH₂O Total
1 7.0 ul - 1.0 ul 2.0 ul 9.17 ul 20.0 ul
2 - 7.0 ul 1.0 ul 2.0 ul 5.0 ul 20.0 ul

Transformation will be made.


Mini Prep of “pSB1C3 – TEV”

# Sample ID User name Date and Time Nucleic Acid Conc. Unit A260 A280 260/280 260/230 Sample Type Factor
1 blank biospec 9/8/2015 10:39:08 AM -0.1 ng/ul -0.003 -0.009 0.30 0.13 DNA 50.00
2 psb1c3-tev 17 biospec 9/8/2015 10:40:15 AM 125.5 ng/ul 2.510 1.331 1.89 2.16 DNA 50.00
3 psb1c3-tev 18 biospec 9/8/2015 10:42:45 AM 111.9 ng/ul 2.238 1.195 1.87 2.14 DNA 50.00


Digestion of “pSB1C3 – TEV 17/18 and pColA-Toehold”

Digestion of “pSB1C3 – TEV 17/18”
DNA (1000 ng/ul) XbaI (FD) SpeI (FD) Fast Digest Buffer ddH₂O Total
17 8.0 ul 1.0 ul 1.0 ul 2.0 ul 8.0 ul 20.0 ul
18 9.0 ul 1.0 ul 1.0 ul 2.0 ul 7.0 ul 20.0 ul

37˚C 1h


Digestion of “pColA-Toehold”
DNA (1000 ng/ul) XbeI (FD) SpeI (FD) Fast Digest Buffer ddH₂O Total
1x 16.0 ul 1.0 ul 1.0 ul 2.0 ul - 20.0 ul

37˚C 1h


Colony PCR of “ pSB1C3-TEV 17/18” (08.09.2015)

Colony PCR
MgCl₂ (NH₄)2SO₄ pTRE Luc fw SV40 rv dNTP Tag ddH₂O DNA Total
1x 2.5 ul 2.5 ul 1.0 ul 1.0 ul 0.5 ul 0.2 ul 12.3 ul 5.0 ul 25.0 ul
25x 61.5 ul 61.5 ul 25.0 ul 25.0 ul 12.5 ul 5.0 ul 307.5 ul 498.0 ul
Cycling
95˚C 95˚C 56˚C 72˚C 72˚C Cycle
Time 5’ 30’’ 30’’ 1.5’ 5’ 35x

Result: negative

GEL GÖRÜNTÜSÜ


Digestion of “pColA / pSB1C3 #9 / pColA-Toehold #2-8 / pSB1C3 T7 #10-17 / #18” (10.11.2015)

Digestion of
DNA (1000 ng/ul) XbaI (FD) SpeI (FD) Cut Smart Buffer ddH₂O Total
pColA 4.5 ul 0.5 ul 0.5 ul 2.0 ul 12.5 ul 20.0 ul
pSB1C3 #9 1.7 ul 0.5 ul 0.5 ul 2.0 ul 15.3 ul 20.0 ul
pColA-Toehold #2-8 16.0 ul 0.5 ul 0.5 ul 2.0 ul 1.0 ul 20.0 ul
pSB1C3 T7 #10-17 3.3 ul 0.5 ul 0.5 ul 2.0 ul 13.7 ul 20.0 ul
#18 3.0 ul 0.5 ul 0.5 ul 2.0 ul 14.0 ul 20.0 ul

37˚C 3h<7p> <p>pColA-Toehold #2-8: 1ul rSAP / 37˚C 1h / 80˚C 20’

Result: pColA and #10 gel extraction will be made.

GEL GÖRÜNTÜLERI


Gel extraction

# Sample ID User name Date and Time Nucleic Acid Conc. Unit A260 A280 260/280 260/230 Sample Type Factor
1 blank biospec 9/10/2015 7:29:27 PM -0.2 ng/ul -0.003 -0.009 0.37 0.28 DNA 50.00
2 psb1c3-#9 biospec 9/10/2015 7:31:01 PM 308.0 ng/ul 6.160 3.182 1.94 2.10 DNA 50.00
# Sample ID User name Date and Time Nucleic Acid Conc. Unit A260 A280 260/280 260/230 Sample Type Factor
1 blank biospec 9/10/2015 8:13:13 PM 0.3 ng/ul 0.005 -0.003 -1.70 -2.25 DNA 50.00
2 psb1c3 t7-10 (17) biospec 9/10/2015 8:14:54 PM 152.2 ng/ul 3.045 1.612 1.89 2.25 DNA 50.00
3 psb1c3 t7-10 (18) biospec 9/10/2015 8:16:02 PM 164.6 ng/ul 3.291 1.737 1.90 2.23 DNA 50.00
# Sample ID User name Date and Time Nucleic Acid Conc. Unit A260 A280 260/280 260/230 Sample Type Factor
1 blank biospec 9/11/2015 10:05:05 PM 0.1 ng/ul 0.002 -0.017 -0.13 -0.11 DNA 50.00
2 pcola (notI) biospec 9/11/2015 10:07:08 PM 40.4 ng/ul 0.807 0.399 2.03 1.75 DNA 50.00
3 psb1c3 #9 biospec 9/11/2015 10:08:33 PM 76.0 ng/ul 1.520 0.789 1.93 2.17 DNA 50.00
4 psb1c3-t7 #10 biospec 9/11/2015 10:09:55 PM 71.5 ng/ul 1.431 0.721 1.99 2.15 DNA 50.00
5 blank biospec 9/11/2015 10:14:04 PM -0.4 ng/ul -0.007 -0.020 0.35 0.32 DNA 50.00
6 psb1c3-t7 #10 biospec 9/11/2015 10:15:01 PM 70.4 ng/ul 1.408 0.727 1.94 2.16 DNA 50.00
# Sample ID User name Date and Time Nucleic Acid Conc. Unit A260 A280 260/280 260/230 Sample Type Factor
1 blank biospec 9/11/2015 2:24:30 AM 0.6 ng/ul 0.011 0.002 4.95 1.18 DNA 50.00
2 Pcola-2 biospec 9/11/2015 2:26:25 AM 145.0 ng/ul 2.901 1.398 2.08 2.04 DNA 50.00