|
|
Line 1,439: |
Line 1,439: |
| </html> | | </html> |
| | | |
− | Colony PCR of „pSB1C3 – Gblocks“ (03.08.2015)
| |
− | {| border="1" class="wikitable"
| |
− | !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
| |
− | |}
| |
| | | |
− | {| 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>Sonuc: negative</p>
| |
− | GEL GÖRÜNTÜSÜ
| |
− |
| |
− | <br>
| |
− |
| |
− | Colony PCR of „pSB1C3 – Gblocks #14“ (03.08.2015)
| |
− | {| border="1" class="wikitable"
| |
− | !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
| |
− | |}
| |
− |
| |
− | {| 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>Sonuc: negative</p>
| |
− | GEL GÖRÜNTÜSÜ
| |
− |
| |
− | <br>
| |
− |
| |
− | Digestion of “pSB1C3-RFP” (03.08.2015)
| |
− | {| border="1" class="wikitable"
| |
− | !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
| |
− | |}
| |
− | <p>37˚C 2h</p>
| |
− | <p>Gel Electropheresis was made.</p>
| |
− | <p>Result: Bands were at the expected section. Gel purification was made.</p>
| |
− |
| |
− | GEL GÖRÜNTÜSÜ
| |
− |
| |
− | <br>
| |
− |
| |
− | Ligation of “pSB1C3 – Gblock #14”
| |
− | {| border="1" class="wikitable"
| |
− | !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
| |
− | |}
| |
− |
| |
− | <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||8/3/2015 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
| |
− | |}
| |
− |
| |
− | <br>
| |
− |
| |
− | PCR of “G-Bloks via Q5 Polymerase” (04.08.2015)
| |
− | {| border="1" class="wikitable"
| |
− | !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
| |
− | |}
| |
− |
| |
− | <br>
| |
− |
| |
− | {| border="1" class="wikitable"
| |
− | !Cycling SV40 rev/CMV fwd
| |
− | |-
| |
− | |||95˚C||95˚C||63˚C||72˚C||72˚C||Cycle
| |
− | |-
| |
− | |Time||5’||30’’||30’’||1’||2’||35x
| |
− | |}
| |
− |
| |
− | <br>
| |
− |
| |
− | {| border="1" class="wikitable"
| |
− | !Cycling CMV fwd/tetR rev
| |
− | |-
| |
− | |||98˚C||98˚C||66˚C||72˚C||72˚C||Cycle
| |
− | |-
| |
− | |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
| |
− | |-
| |
− | |1x||2.0 ul||2.0 ul||1.0 ul||1.0 ul||14.0 ul||20.0 ul
| |
− | |}
| |
− |
| |
− | <p>37˚C 4h</p>
| |
− | <p>Result: Bands were at the expected section.</p>
| |
− | GEL GÖRÜNTÜSÜ
| |
− |
| |
− | <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||8/4/2015 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
| |
− | |}
| |
− |
| |
− | <br>
| |
− |
| |
− | Colony PCR of “11,12,13 pSB1C3 - GBlocks” (04.08.2015)
| |
− | {| border="1" class="wikitable"
| |
− | !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||
| |
− | |}
| |
− |
| |
− | {| 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
| |
− | |}
| |
− |
| |
− | <p>Result: negative</p>
| |
− | GEL GÖRÜNTÜSÜ
| |
− |
| |
− | <br>
| |
− |
| |
− | Ligation and Transformation of “5,6,7,8,9 pSB1C3-GBlocks” (04.08.2015)
| |
− | {| border="1" class="wikitable"
| |
− | !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
| |
− | |}
| |
− | <p>50ng DNA</p>
| |
− | <p>1 vector:0.5 insert</p>
| |
− | <p>Transfomation with TOP10.</p>
| |
− |
| |
− | <br>
| |
− |
| |
− | Ligation of “GBlocks 11-12 pSB1C3” (05.08.2015)
| |
− | {| border="1" class="wikitable"
| |
− | !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.
| |
− |
| |
− | <br>
| |
− |
| |
− | Colony PCR of “5,6,7,8,9,T7 system – Gblocks” (05.08.2015)
| |
− | {| border="1" class="wikitable"
| |
− | !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
| |
− | |}
| |
− | {| border="1" class="sortable"
| |
− | !Cycling
| |
− | |-
| |
− | |||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle
| |
− | |-
| |
− | |Time||5’||30’’||30’’||1.5’||5’||35x
| |
− | |}
| |
− | GEL GÖRÜNTÜSÜ
| |
− |
| |
− | <br>
| |
− |
| |
− | Ligation of “GBlocks 2,5,6,7,8,9,10,12,13,15,17 pSB1C3-RFP” (06.08.2015)
| |
− | {| border="1" class="wikitable"
| |
− | !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.
| |
− |
| |
− | <br>
| |
− |
| |
− |
| |
− | Colony PCR of “pTRE-1,13,15” (06.08.2015)
| |
− | {| border="1" class="wikitable"
| |
− | !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
| |
− | |}
| |
− | {| 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 “5,6,7,9 – Gblocks” (06.08.2015)
| |
− | {| border="1" class="wikitable"
| |
− | !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
| |
− | |}
| |
− |
| |
− | {| 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 “7,8,10 – Gblocks” from Masterplate (08.08.2015)
| |
− | {| border="1" class="wikitable"
| |
− | !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
| |
− | |}
| |
− | {| 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 “pET45” (09.08.2015)
| |
− | {| border="1" class="wikitable"
| |
− | !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
| |
− | |}
| |
− | <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
| |
− | |-
| |
− | |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
| |
− | |}
| |
− |
| |
− |
| |
− | <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
| |
− | |-
| |
− | |||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
| |
− | |}
| |
− | <p>16˚C 2h</p>
| |
− |
| |
− | <br>
| |
− |
| |
− | Digestion of “pSB1C3 – HNS”
| |
− | {| border="1" class="wikitable"
| |
− | !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
| |
− |
| |
− | <br>
| |
− |
| |
− | Colony PCR of “pTRE #13”
| |
− | {| border="1" class="wikitable"
| |
− | !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
| |
− | |}
| |
− | {| border="1" class="wikitable"
| |
− | !Cycling
| |
− | |-
| |
− | |||95˚C||95˚C||60˚C||72˚C||72˚C||Cycle
| |
− | |-
| |
− | |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
| |
− | |-
| |
− | |||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
| |
− | |}
| |
− | {| 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 GÖRÜNTÜLERI
| |
− |
| |
− | <br>
| |
− |
| |
− | Digestion of „pColA-KanR and „pSB1C3-Toehold“ (10.08.2015)
| |
− | {| border="1" class="wikitable"
| |
− | !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||||||
| |
− | |}
| |
− |
| |
− | <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"
| |
− | !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
| |
− | |-
| |
− | |-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"
| |
− | !#!!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
| |
− | |}
| |
− | <p>Result: The Band of pColA were at the expected section. 2000 pb</p>
| |
− | (CUT CHECK GÖRÜNTÜSÜ)(12.08.2015)
| |
− |
| |
− | <br>
| |
− |
| |
− | Ligation of “#2 GBlock with pET45” (11.08.2015)
| |
− | {| border="1" class="wikitable"
| |
− | !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
| |
− | |}
| |
− | <p>22˚C - 1’40’’</p>
| |
− | Transformation was made.
| |
− |
| |
− | <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||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
| |
− | |}
| |
− |
| |
− | <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||8/12/2015 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
| |
− | |}
| |
− |
| |
− | <br>
| |
− |
| |
− | Cut Check of “pET45 1,4,5,6,7,8,9,10 – BamHI/XhoI” (13.08.2015)
| |
− | {| border="1" class="wikitable"
| |
− | !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
| |
− | |}
| |
− | <p>Dogru cikan koloniler BL21 bakterisine transformasyon edildi ve sivi kültür yapildi. Sonrasinda protein izolasyon ve WB yapilacak.</p>
| |
− |
| |
− | GEL GÖRÜNTÜLERI
| |
− |
| |
− | <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||8/13/2015 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
| |
− | |}
| |
− |
| |
− | <br>
| |
− |
| |
− | Colony PCR of “pTRE – 12,13 GBlocks” (14.08.2015)
| |
− | {| border="1" class="wikitable"
| |
− | !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
| |
− | |}
| |
− | {| 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>Sonuc: negative</p>
| |
− |
| |
− | GEL GÖRÜNTÜSÜ
| |
− |
| |
− | <br>
| |
− |
| |
− | Colony PCR of “pColA – Toehold GFP” (14.08.2015)
| |
− | {| 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
| |
− | |-
| |
− | |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: 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"
| |
− | !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
| |
− | |}
| |
− | <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"
| |
− | !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
| |
− | |}
| |
− | {| border="1" class="wikitable"
| |
− | !Cycling
| |
− | |-
| |
− | |||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle
| |
− | |-
| |
− | |Time||5’||30’’||30’’||1.5’||5’||35x
| |
− | |}
| |
− | GEL GÖRÜNTÜLERI
| |
− |
| |
− | <br>
| |
− |
| |
− | Digestion/Gel Electrophoresis/Gel Extraction and Purification of “pSB1C3” (16.08.2015)
| |
− | {| border="1" class="wikitable"
| |
− | !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
| |
− | |}
| |
− | <p>37˚C 1h</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||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
| |
− | |}
| |
− |
| |
− | <br>
| |
− |
| |
− | Colony PCR of “pSB1C3 7,10,12,13 and pET45 2” (14.08.2015)
| |
− | {| border="1" class="wikitable"
| |
− | !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
| |
− | |}
| |
− | {| 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>
| |
− |
| |
− | {| border="1" class="wikitable"
| |
− | !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
| |
− | |}
| |
− | {| 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>
| |
− |
| |
− | {| 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||8/16/2015 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
| |
− | |}
| |
− |
| |
− | <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>
| |
− | Ligation of “pColA and pSB1C3 #9 (Damp-Pex)” (18.08.2015)
| |
− | {| border="1" class="wikitable"
| |
− | !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
| |
− | |}
| |
− |
| |
− | <br>
| |
− |
| |
− | Miniprep of “pTet-off DH5α /Top10/NEB5fα/NEB10β”
| |
− | {| 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||8/18/2015 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
| |
− | |}
| |
− |
| |
− | <br>
| |
− |
| |
− | Colony PCR of “pET45 #2.8” (15.08.2015)
| |
− | {| border="1" class="wikitable"
| |
− | !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
| |
− | |}
| |
− | {| 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 17,6,8” (S. 118/15.08.2015)
| |
− | {| border="1" class="wikitable"
| |
− | !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
| |
− | |}
| |
− | {| 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: 6: Liquid Culture was made.</p>
| |
− | <p>8: sequencing will be done</p>
| |
− | <p>17: Colony PCR will be repeated.</p>
| |
− | GEL GÖRÜNTÜSÜ
| |
− |
| |
− | <br>
| |
− |
| |
− | Colony PCR of “pColA blunt”
| |
− |
| |
− | {| border="1" class="wikitable"
| |
− | !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
| |
− | |}
| |
− | {| 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>
| |
− |
| |
− | PCR of “Q5 DNA Polymerase for pCAG” (19.08.2015)
| |
− | {| border="1" class="wikitable"
| |
− | !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
| |
− | |}
| |
− | {| border="1" class="wikitable"
| |
− | !Cycling
| |
− | |-
| |
− | |||98˚C||98˚C||70˚C||72˚C||72˚C||Cycle
| |
− | |-
| |
− | |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
| |
− | |-
| |
− | |||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
| |
− | |}
| |
− | <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
| |
− | |-
| |
− | |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
| |
− | |}
| |
− |
| |
− | <br>
| |
− |
| |
− | Digestion of “pSB1C3 – mLacI miR373-BS” (19.08.2015)
| |
− | {| border="1" class="sortable"
| |
− | !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||
| |
− | |}
| |
− | <p>37˚C 2.30h</p>
| |
− |
| |
− | <br>
| |
− |
| |
− | Ligation of “pSB1C3 – mLacI miR373-BS with pTRE”
| |
− | {| border="1" class="wikitable"
| |
− | !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
| |
− | |}
| |
− | <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
| |
− | |-
| |
− | |||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
| |
− | |}
| |
− | {| 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. Liquid Culture was made.</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||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
| |
− | |}
| |
− |
| |
− | <br>
| |
− |
| |
− | Cut Check of “#6,8”
| |
− | {| border="1" class="wikitable"
| |
− | !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
| |
− | |}
| |
− | <p>37˚C 40’</p>
| |
− | <p>Result: positive</p>
| |
− | GEL GÖRÜNTÜSÜ
| |
− |
| |
− | <br>
| |
− |
| |
− | Colony PCR “pSB1C3 17,6,8 – pColA, Damp-Pex, pTetoff” (19.08.2015)
| |
− |
| |
− | GEL GÖRÜNTÜLERI
| |
− |
| |
− | <br>
| |
− |
| |
− | Mini Prep of “pSB1C3-#17”
| |
− | {| 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||8/20/2015 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
| |
− | |}
| |
− |
| |
− | <br>
| |
− |
| |
− | 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>
| |
− |
| |
− | {| border="1" class="wikitable"
| |
− | !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||
| |
− | |}
| |
− | <p>1ul rSAP / 37˚C 1h</p>
| |
− |
| |
− | <br>
| |
− |
| |
− | {| border="1" class="wikitable"
| |
− | !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||
| |
− | |}
| |
− | <p>22˚C 2h</p>
| |
− | <p>-80 ˚C 20’’ Heat Inaktivation</p>
| |
− |
| |
− | <br>
| |
− |
| |
− | Colony PCR of “pTRE - #13 mLacI-miR373 BS” (21.08.2015)
| |
− | {| border="1" class="wikitable"
| |
− | !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
| |
− | |}
| |
− | {| 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>Results: negative</p>
| |
− | GEL GÖRÜNTÜLERI
| |
− |
| |
− | <br>
| |
− |
| |
− | Mini Prep of “pSB1C3 #6-25,26”
| |
− | {| 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||8/20/2015 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
| |
− | |}
| |
− |
| |
− | <br>
| |
− |
| |
− | Cut Check of “pSB1C3 #6-25,26”
| |
− | {| border="1" class="wikitable"
| |
− | !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
| |
− | |}
| |
− | <p>37˚C 3h</p>
| |
− | <p>Result: negative</p>
| |
− |
| |
− | <br>
| |
− |
| |
− | Digestion of “pTRE #12”
| |
− | {| border="1" class="wikitable"
| |
− | !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
| |
− | |}
| |
− | <p>37˚C 3h</p>
| |
− | <p>Result: negative</p>
| |
− | GEL GÖRÜNTÜSÜ
| |
− |
| |
− | <br>
| |
− |
| |
− | Digestion of “Damp-Pex pSB1C3” (22.08.2015)
| |
− | {| border="1" class="wikitable"
| |
− | !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||||
| |
− | |}
| |
− | <p>37˚C 3h</p>
| |
− |
| |
− | <br>
| |
− |
| |
− | Ligation of “Damp-Pex pColA”
| |
− | {| border="1" class="wikitable"
| |
− | !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||
| |
− | |}
| |
− | <p>22˚C 1.30h</p>
| |
− | <p>Transformation with NEB5α.</p>
| |
− |
| |
− | <br>
| |
− |
| |
− | Colony PCR of “Damp-Pex + pColA” (23.08.2015)
| |
− | {| 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
| |
− | |-
| |
− | |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: #5-4, #5-9 Liquid Culture was made.</p>
| |
− | GEL GÖRÜNTÜSÜ
| |
− |
| |
− | <br>
| |
− |
| |
− | Mini Prep of “pTetoff #14-4,5,8” (22.08.2015)
| |
− | {| 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||8/22/2015 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
| |
− | |}
| |
− |
| |
− | <br>
| |
− |
| |
− | {| border="1" class="wikitable"
| |
− | !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
| |
− | |}
| |
− | <p>37˚C 2h</p>
| |
− | <p>Result: negative</p>
| |
− |
| |
− | GEL GÖRÜNTÜSÜ
| |
− |
| |
− | <br>
| |
− |
| |
− | {| border="1" class="wikitable"
| |
− | !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
| |
− | |}
| |
− | <p>37˚C 30’’</p>
| |
− | <p>Result: negative</p>
| |
− | GEL GÖRÜNTÜSÜ
| |
− |
| |
− | <br>
| |
− |
| |
− | Colony PCR of “pSB1C3 – T7 #4,5,6,8” (23.08.2015)
| |
− | {| 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
| |
− | |-
| |
− | |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"
| |
− | !Cycling
| |
− | |-
| |
− | |||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle
| |
− | |-
| |
− | |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>
| |
− |
| |
− | <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||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
| |
− | |}
| |
− |
| |
− | <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>
| |
− |
| |
− | Ligation of “pCAGop with pTRE #12-2” (24.08.2015)
| |
− | {| 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>
| |
− |
| |
− | <br>
| |
− |
| |
− | 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>
| |
− |
| |
− | {| 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||8/25/2015 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
| |
− | |}
| |
− |
| |
− | <br>
| |
− |
| |
− | Colony PCR of “pTRE #13” (25.08.2015)
| |
− | {| border="1" class="wikitable"
| |
− | !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
| |
− | |}
| |
− | {| border="1" class="wikitable"
| |
− | !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
| |
− |
| |
− | <br>
| |
− |
| |
− | Colony PCR of “pCAG-DsRed” (25.08.2015)
| |
− | {| 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: Bands weren’t at the expected section.</p>
| |
− | GEL GÖRÜNTÜSÜ
| |
− |
| |
− | <br>
| |
− |
| |
− | Cut Check of „pSB1C3 – TlpB“ (25.05.2015)
| |
− | {| border="1" class="wikitable"
| |
− | !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||
| |
− | |}
| |
− | <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
| |
− | |-
| |
− | |||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||||
| |
− | |}
| |
− | <p>37˚C 3h</p>
| |
− |
| |
− | <br>
| |
− |
| |
− | {| border="1" class="wikitable"
| |
− | !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.
| |
− |
| |
− | <br>
| |
− |
| |
− | Colony PCR of “pCAGop-DsRed” (26.08.2015)
| |
− | {| 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
| |
− | |-
| |
− | |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: 2 colonie will be made liquid culture and streak plate. Ligation and transformation will be repeated.</p>
| |
− | GEL GÖRÜNTÜSÜ
| |
− |
| |
− | <br>
| |
− |
| |
− | 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>
| |
− |
| |
− | </section>
| |
− |
| |
− | </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
| |
− | |}
| |
| | | |
| | | |