Team:DTU-Denmark/Notebook/23 August 2013
From 2013.igem.org
23 August 2013
Contents |
Lab 208
Main purpose
- PCR to isolate Biobricks
- PCR to make the constitutive reference promoter
- colony PCR on Nir transformants
Who was in the lab
Kristian, Henrike
Procedure
PCR Biobrick Isolation
Used new primers to isolate Hello World constructs. Used HF buffer and tested three different concentrations of DMSO: 0%, 2% and 5%.
Primers: 56a, 56b
template: TAT2-1 (miniprep)
program:
temperature | time | cycles |
---|---|---|
98C | 2:00 | - |
98C | 0:20 | 36 |
56C | 0:30 | 36 |
72C | 0:45 | 36 |
72C | 5:00 | - |
10C | hold | - |
PCR constitutive reference promoter
Got new primers for the constitutive reference promoter since the old reverse primer (52b2) had a mistake. Repeated reaction set-up and program from the 13.08. Used GC buffer, 5% DMSO and 2uL 50 mM MgCl2.
primers: 52a, 52bn
template: pZA21::RFP
program:
temperature | time | cycles |
---|---|---|
98C | 2:00 | - |
98C | 0:20 | 36 |
58.1C | 1:00 | 36 |
72C | 3:00 | 36 |
72C | 5:00 | - |
10C | hold | - |
Colony PCR on Nir transformants
Used Q5 premix from NEB.
Reagent | volume (in uL) |
---|---|
Q5 master mix | 12,5 |
FW primer | 3 |
RV primer | 3 |
template | 1 |
MilliQ | 5,5 |
primers: 45a, 45b
program:
temperature | time | cycles |
---|---|---|
98C | 10:00 | - |
98C | 0:10 | 36 |
56C | 0:30 | 36 |
72C | 0:45 | 36 |
72C | 5:00 | - |
10C | hold | - |
Results
Gel electrophoresis
- 1 kb ladder
- Sec2 Biobrick
- Sec2 Biobrick
- Sec2 neg
- col AMO 1
- col AMO 2
- col AMO 3
- col AMO 4
- col AMO 5
- col AMO 6
- col AMO 7
- col AMO 8
- col AMO 9
- col AMO 10
- 1 kb ladder
- 1 kb ladder
- col Nir
- constitutive reference promoter
- constitutive reference promoter
- Biobrick Isolation TAT2-1 negative
- Biobrick Isolation TAT2-1 2% DMSO
- Biobrick Isolation TAT2-1 5% DMSO
- Biobrick Isolation TAT2-1 w/o DMSO
- 1 kb ladder
Conclusion
The transformation of AMO into pZA21::ara was successful, all tested colonies have the insert.
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