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

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Creating “pCAG (Plasmid) – Repeat (23.07.2015)
 
Creating “pCAG (Plasmid) – Repeat (23.07.2015)
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{| border="1" class="wikitable"
 
!PCR from “pCAGGS”
 
!PCR from “pCAGGS”
 
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|-
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{| border="1" class="wikitable"
 
!Cycling
 
!Cycling
 
|-
 
|-
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<br>
 
<br>
  
−
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{| 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
 
|-
 
|-
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|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
 
|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
 
|}
 
|}
 +
 +
<br>
 +
 +
Resuspension of “Newly Arrived G-Blocks from IDT” (S. 23/23.7.2015)
 +
<p>100 ul TE for all tubes.</p>
 +
{| border="1" class="wikitable"
 +
!!!!!ng!!fmol!!TE ul!!fmol/ul!!ul of inserts for 75 fmol
 +
|-
 +
|1||Toehold for cola||1000||1523||100||15.23||4.92449
 +
|-
 +
|2||TnrA-pTnrA-RFP||1000||1032||100||10.32||7.26744
 +
|-
 +
|3||ColA-KanR-dTer||1000||816||100||8.16||9.19118
 +
|-
 +
|4||HNS for PET||500||1578||100||15.78||4.75285
 +
|-
 +
|5||HNS-T108I for PET||500||1578||100||15.78||4.75285
 +
|-
 +
|6||potB59-pomA for PET ||1000||892||100||8.92||8.40807
 +
|-
 +
|7||Gad E – for PET||500||1291||100||12.91||5.80945
 +
|-
 +
|8||TlpB for PET||1000||901||100||9.01||8.32408
 +
|-
 +
|9||DAMP-Pex for PET/pcolA||1000||2089||100||20.89||3.59023
 +
|-
 +
|10||Tev Protease for PET||1000||1948||100||19.48||3.8501
 +
|-
 +
|11||miRNA switch- miR373- BS for pTET||500||2212||100||22.12||3.3906
 +
|-
 +
|12||LacO- DsRed- miR26a-375 pC||1000||1709||100||17.09||4.38853
 +
|-
 +
|13||mLacI-miR373 BS for pTRE||1000||1280||100||12.8||5.85938
 +
|-
 +
|14||miRNA switch- miR 21 BS- miR 223||1000||1902||100||19.02||3.94322
 +
|-
 +
|15||mLacI-miR223 BS miR 21 BS for pTRE||1000||1272||100||12.72||5.89623
 +
|-
 +
|16||Trigger RNA for pSB1C3||250||1910||100||19.1||3.9267
 +
|-
 +
|17||PsicA for pSB1C3||1000||1546||100||15.46||4.86
 +
|-
 +
|18||MVF-sicA for ColA||1000||1042||100||10.42||7.20
 +
|}
 +
 +
Not: 100ng pSB1C3 (2050 bp) ≈ 75 fmol
 +
 +
<br>
 +
 +
Digestion of „G-Blocks from IDT and pSB1C3“ (23.07.2015)
 +
{| border="1" class="wikitable"
 +
!Digestion
 +
|-
 +
|||Insert ||EcoRI-HF||PstI||NEB 2.1 Buffer||ddH₂O||Total
 +
|-
 +
|1x||10.0 ul||1.0 ul||1.0 ul||2.0 ul||6.0 ul||20.0 ul
 +
|-
 +
|17x||10.0 ul||17.0 ul||17.0 ul||34.0 ul||102.0 ul||20.0 ul
 +
|}
 +
 +
{| border="1" class="wikitable"
 +
!Digestion
 +
|-
 +
|||Vector||EcoRI-HF||PstI||NEB 2.1 Buffer||ddH₂O||Total
 +
|-
 +
|2x||17.0 ul||1.0 ul||1.0 ul||2.0 ul||-||21.0 ul
 +
|}
 +
<p>Result: Gel extraction was made.</p>
 +
GEL GÖRÜNTÜSÜ
 +
 +
<br>
 +
  
  

Revision as of 10:36, 14 September 2015


NOTEBOOK

December

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January

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February

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March

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April

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May

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June

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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.

GEL GÖRÜNTÜSÜ


(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.

GEL GÖRÜNTÜSÜ


July

Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description..

Gradient PCR of „pCAG with CAG FWD/CAG REV. Primers/pCMV REV.” (01.07.2015)

Gradient PCR from pCAGGS (CAG FWD – CAG REVERSE)
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

60-68˚C

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

GEL GÖRÜNTÜSÜ


Gradient PCR from pCAGGS (CAG FWD – Chicken β Aktin REVERSE)
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

60-68˚C

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

GEL GÖRÜNTÜSÜ


Protocols of „Phusion Pol, and Q5 Polymerase” (02.07.2015)

Phusion DNA Polymerase
dNTP Fwd primer Rev primer Buffer Phusion Pol. 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
Cycling
98˚C 98˚C ???˚C 72˚C 72˚C Cycle
Time 2’ 10’’ 30’’ 1’ 5’ ???x


Q5 DNA Polymerase
dNTP Fwd primer Rev primer Buffer Q5 Pol. ddH₂O DNA Total
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
Cycling
98˚C 98˚C ???˚C 72˚C 72˚C Cycle
Time 2’ 10’’ 30’’ 1’ 5’ ???x

Defterde jel görüntüsü yok.

Gradient PCR of „pCAG with CAG-FWD/CAG REV. Primers and Phusion Pol.” (07.07.2015)

Gradient PCR from pCAGGS
dNTP Fwd primer Rev primer ?????
Buffer Phusion Pol. 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

62-64˚C

Result: Gel extraction was performed. PCR (+): 680 bp

GEL GÖRÜNTÜSÜ


# Sample ID User name Date and Time Nucleic Acid Conc. Unit A260 A280 260/280 260/230 Sample Type Factor
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
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


Creating “Plasmid pCAG” via “pTRE” and “Promoter pCAG” (09.07.2015)

Digestion of “pTRE” and “Promoter pCAG”
pTRE (1536 ng/ul) pCAG (promoter) (13.7 ng/ul) EcoRI XhoI Neb 3.1 Buffer ddH₂O Total
1 3.2 ul - 0.5 ul 0.5 ul 2.0 ul 13.8 ul 20.0 ul
2 - 10.0 ul 0.5 ul 0.5 ul 2.0 ul 7.0 ul 20.0 ul

pTRE-delta-TRE was made after digestion.

Concentration: 99.9 ng/ul

The final concentration of pCAG is 6.855 ng/ul.


Ligation of „pTRE TRE“ and „Digested promoter pCAG“
pTRE TRE
pCAG T4 DNA Ligase Buffer ddH₂O Total
1:1 3.0 ul 7.0 ul 0.5 ul 2.0 ul 7.5 ul 20.0 ul

Ligation products were transformed into E.Coli/BL321 strain.

Result: No colonies were observed.


Digestion of “pTEToff and pET45 Vectors” (10.07.2015)

Digestion of “pTEToff and pET45 Vectors”
pTEToff (1141 ng/ul) pET45 (485 ng/ul) XhoI (Neb) BamHI (Neb) SalI (Thermo) HindIII (Thermo) Cut Smart Buffer Fast Digest Buffer ddH₂O Total
1 3.1 ul - - - 0.5 ul 0.5 ul - 2.0 ul 13.9 ul 20.0 ul 37˚C 1h
2 - 4.1 ul 0.5 ul 0.5 ul - - 2.0 ul - 12.9 ul 20.0 ul 37˚C 2h

Result: Bands were at the expected section. 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 eb biospec 7/10/2015 11:37:54 PM -0.4 ng/ul -0.008 -0.015 0.58 -0.39 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
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


Creating “Plasmid pCAG” via “pTRE” and “Promoter pCAG” – Repeat (08.07.2015)

Digestion of “pTRE with EcoRI/XhoI”
pTRE XhoI EcoRI Neb 2.1 Buffer ddH₂O Total
1 3.2 ul 0.5 ul 0.5 ul 2.0 ul 13,8 ul 20.0 ul 37˚C 2h
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’

Result: 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 eb biospec 7/9/2015 6:32:17 PM -0.7 ng/ul -0.015 -0.022 0.66 0.21 DNA 50.00
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
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
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


Creating “Plasmid pCAG” – Continue (12.07.2015)

Ligation of „pTRE TRE“ and „Digested promoter pCAG“
pTRE TRE CIP (+) pTRE TRE CIP (-) pCAG (6.85 ng/ul) T4 DNA Ligase Buffer ddH₂O Total
1 3.0 ul - 7.0 ul 0.5 ul 2.0 ul 7.5 ul 20.0 ul
2 - 3.0 ul 7.0 ul 0.5 ul 2.0 ul 7.5 ul 20.0 ul

Room Temperature 1h

Sonuc: Transformation was made. No colonies were observed at first plate. At the second plate there was five colonies. Colony PCR will be made.


Creating “Plasmid pCAG” – Continue (13.07.2015)

Colony PCR from “pCAG” with “pTRE Luc fwd/SV40 polyA re. primers”
MgCl₂ (NH₄)2SO₄ pTRE Luc fwd 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
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 55˚C 72˚C 72˚C Cycle
Time 5’ 30’’ 30’’ 1.5’ 5’ 35x

Result: All bands were observed around 700 bp, results were negative. Ligation will be repeated.

(+) bant: 923 bp

(-) bant: 705 bp

GEL GÖRÜNTÜSÜ


Creating “Plasmid pCAG” – Repeat (19.07.2015)

Ligation of „pTRE TRE“ and „Digested Promoter pCAG“
pTRE TRE CIP (+) pTRE TRE CIP (-) pCAG (6.85 ng/ul) T4 DNA Ligase Buffer ddH₂O Total
1 3.0 ul - 2.5 ul 0.5 ul 2.0 ul 12.0 ul 20.0 ul
2 - 3.0 ul 2.5 ul 0.5 ul 2.0 ul 12.0 ul 20.0 ul

Vector:Insert

3:1

RT 2h

Transformation at BL21.

CIP (+): No colonies were absorved.

CIP (-): Colony PCR will be made.


Creating “Plasmid pCAG” – Continue (20.07.2015)

Colony PCR from “pCAG” with “pTRE Luc fwd/SV40 polyA re. primers”
MgCl₂ (NH₄)2SO₄ pTRE Luc fwd 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
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 55˚C 72˚C 72˚C Cycle
Time 5’ 30’’ 30’’ 1.5’ 5’ 35x

Result: All bands were observed around 700 bp, results were negative. Ligation will be repeated.

(+) bant: 923 bp

(-) bant: 705 bp

GEL GÖRÜNTÜSÜ


Creating “pCAG (Plasmid) – Repeat (23.07.2015)

PCR from “pCAGGS”
dNTP CAG fwd CAG rev Chicken β Akt. Rev pCAGGS Phusion Pol 6C??
GC??
Buffer ddH₂O 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
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
Cycling
98˚C 98˚C 64/68˚C 72˚C 72˚C Cycle
Time 2’ 10’’ 30’’ 1’ 5’ 35x

Gel Extraction will made.

GEL GÖRÜNTÜSÜ


# Sample ID User name Date and Time Nucleic Acid Conc. Unit A260 A280 260/280 260/230 Sample Type Factor
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
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
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


Resuspension of “Newly Arrived G-Blocks from IDT” (S. 23/23.7.2015)

100 ul TE for all tubes.

ng fmol TE ul fmol/ul ul of inserts for 75 fmol
1 Toehold for cola 1000 1523 100 15.23 4.92449
2 TnrA-pTnrA-RFP 1000 1032 100 10.32 7.26744
3 ColA-KanR-dTer 1000 816 100 8.16 9.19118
4 HNS for PET 500 1578 100 15.78 4.75285
5 HNS-T108I for PET 500 1578 100 15.78 4.75285
6 potB59-pomA for PET 1000 892 100 8.92 8.40807
7 Gad E – for PET 500 1291 100 12.91 5.80945
8 TlpB for PET 1000 901 100 9.01 8.32408
9 DAMP-Pex for PET/pcolA 1000 2089 100 20.89 3.59023
10 Tev Protease for PET 1000 1948 100 19.48 3.8501
11 miRNA switch- miR373- BS for pTET 500 2212 100 22.12 3.3906
12 LacO- DsRed- miR26a-375 pC 1000 1709 100 17.09 4.38853
13 mLacI-miR373 BS for pTRE 1000 1280 100 12.8 5.85938
14 miRNA switch- miR 21 BS- miR 223 1000 1902 100 19.02 3.94322
15 mLacI-miR223 BS miR 21 BS for pTRE 1000 1272 100 12.72 5.89623
16 Trigger RNA for pSB1C3 250 1910 100 19.1 3.9267
17 PsicA for pSB1C3 1000 1546 100 15.46 4.86
18 MVF-sicA for ColA 1000 1042 100 10.42 7.20

Not: 100ng pSB1C3 (2050 bp) ≈ 75 fmol


Digestion of „G-Blocks from IDT and pSB1C3“ (23.07.2015)

Digestion
Insert EcoRI-HF PstI NEB 2.1 Buffer ddH₂O Total
1x 10.0 ul 1.0 ul 1.0 ul 2.0 ul 6.0 ul 20.0 ul
17x 10.0 ul 17.0 ul 17.0 ul 34.0 ul 102.0 ul 20.0 ul
Digestion
Vector EcoRI-HF PstI NEB 2.1 Buffer ddH₂O Total
2x 17.0 ul 1.0 ul 1.0 ul 2.0 ul - 21.0 ul

Result: Gel extraction was made.

GEL GÖRÜNTÜSÜ




August

Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description..

Background of acid resistance.. Background of acid resistance.. Background of acid resistance.. Background of acid resistance.. Background of acid resistance.. Background of acid resistance..

Design of acid resistance.. Design of acid resistance.. Design of acid resistance.. Design of acid resistance.. Design of acid resistance.. Design of acid resistance.. Design of acid resistance..

Results of acid resistance.. Results of acid resistance.. Results of acid resistance.. Results of acid resistance.. Results of acid resistance.. Results of acid resistance.. Results of acid resistance..

Results of acid resistance.. Results of acid resistance.. Results of acid resistance.. Results of acid resistance.. Results of acid resistance.. Results of acid resistance.. Results of acid resistance..

September

Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description..

Background of acid resistance.. Background of acid resistance.. Background of acid resistance.. Background of acid resistance.. Background of acid resistance.. Background of acid resistance..

Design of acid resistance.. Design of acid resistance.. Design of acid resistance.. Design of acid resistance.. Design of acid resistance.. Design of acid resistance.. Design of acid resistance..

Results of acid resistance.. Results of acid resistance.. Results of acid resistance.. Results of acid resistance.. Results of acid resistance.. Results of acid resistance.. Results of acid resistance..

Results of acid resistance.. Results of acid resistance.. Results of acid resistance.. Results of acid resistance.. Results of acid resistance.. Results of acid resistance.. Results of acid resistance..