Team:DTU-Denmark/Notebook/14 August 2013
From 2013.igem.org
14 August 2013
Contents |
lab 208
Main purpose
- PCR for extraction of Nir2 from PAO1
- extract fragments for SPL and perform USER ligation into pZA21::RFP without promoter
Who was in the lab
Ariadni, Helen, Natalia
Procedure
PCR for Nir2 extraction from PAO1
We tried to reproduce the successful extraction of Nir2 from PAO1. The extracted Nir2 fragment will be used as template for a screening PCR with USER primers.
primers: 42a and 42b
template: 1 colony of P. aeruginosa cells resuspended in 100 uL milliQ water
program: touchdown PCR (called U2 on Eppendorf)
The table shows the composition of each reaction:
component (per reaction) | without additives | 5% DMSO | 2mM MgCl2 | 5% DMSO + 2mM MgCl2 | Negative control |
---|---|---|---|---|---|
dNTPs | 1uL | 1uL | 1uL | 1uL | 1uL |
GC buffer | 10uL | 10uL | 10uL | 10uL | 10uL |
Phusion polymerase | 0.5uL | 0.5uL | 0.5uL | 0.5uL | 0.5uL |
MilliQ water | 31.5uL | 29uL | 29.5uL | 27uL | 32.5uL |
template | 1uL | 1uL | 1uL | 1uL | - |
FW primer | 3uL | 3uL | 3uL | 3uL | 3uL |
RV primer | 3uL | 3uL | 3uL | 3uL | 3uL |
DMSO | - | 2.5uL | - | 2.5uL | - |
MgCl2 | - | - | 2uL | 2uL | - |
PCR for Nir2 with USER endings
We are still trying to amplify Nir2 with endings that are suitable for USER cloning with Nir1. The same reaction mix as yesterday was run on two new programs.
Sample 1-6: 63C annealing temperature
Sample 7-12: ramp from 65C-57C
If this PCR is not successful we suggest to try touchdown.
Gel purification
gel purify PCR products from constitutive promoter SPL and constitutive promoter ref
Results
Purification gel
- 1 kb ladder
- constitutive promoter SPL
- constitutive promoter SPL
- constitutive promoter reference
- constitutive promoter reference
- araBAD promoter reference
- araBAD promoter reference
- 1 kb ladder
Conclusion
lab 115
Main purpose
- Find the proper amount of glucose substrate for DM minimal medium
Who was in the lab
Gosia, Kristian, Julia, Henrike
Procedure
PCR for Nir2 extraction from PAO1
We tried to reproduce the successful extraction of Nir2 from PAO1. The extracted Nir2 fragment will be used as template for a screening PCR with USER primers.
primers: 42a and 42b
template: 1 colony of P. aeruginosa cells resuspended in 100 uL milliQ water
program: touchdown PCR (called U2 on Eppendorf)
The table shows the composition of each reaction:
component (per reaction) | without additives | 5% DMSO | 2mM MgCl2 | 5% DMSO + 2mM MgCl2 | Negative control |
---|---|---|---|---|---|
dNTPs | 1uL | 1uL | 1uL | 1uL | 1uL |
GC buffer | 10uL | 10uL | 10uL | 10uL | 10uL |
Phusion polymerase | 0.5uL | 0.5uL | 0.5uL | 0.5uL | 0.5uL |
MilliQ water | 31.5uL | 29uL | 29.5uL | 27uL | 32.5uL |
template | 1uL | 1uL | 1uL | 1uL | - |
FW primer | 3uL | 3uL | 3uL | 3uL | 3uL |
RV primer | 3uL | 3uL | 3uL | 3uL | 3uL |
DMSO | - | 2.5uL | - | 2.5uL | - |
MgCl2 | - | - | 2uL | 2uL | - |
PCR for Nir2 with USER endings
We are still trying to amplify Nir2 with endings that are suitable for USER cloning with Nir1. The same reaction mix as yesterday was run on two new programs.
Sample 1-6: 63C annealing temperature
Sample 7-12: ramp from 65C-57C
If this PCR is not successful we suggest to try touchdown.
Gel purification
gel purify PCR products from constitutive promoter SPL and constitutive promoter ref
Results
Purification gel
- 1 kb ladder
- constitutive promoter SPL
- constitutive promoter SPL
- constitutive promoter reference
- constitutive promoter reference
- araBAD promoter reference
- araBAD promoter reference
- 1 kb ladder
Conclusion
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