"
Team:DTU-Denmark/Notebook/14 August 2013
From 2013.igem.org
(Difference between revisions)
(→PCR for Nir2 extraction from PAO1) |
(→Procedure) |
||
| Line 15: | Line 15: | ||
===PCR for Nir2 extraction from PAO1=== | ===PCR for Nir2 extraction from PAO1=== | ||
| - | + | We tried to reproduce the successive extraction of Nir2 from PAO1. | |
The extracted Nir2 fragment will be used as template for a screening PCR with USER primers | The extracted Nir2 fragment will be used as template for a screening PCR with USER primers | ||
primers: 42a and 42b | primers: 42a and 42b | ||
| + | |||
template: 1 colony of P. aeruginosa cells resuspended in 100 uL milliQ water | template: 1 colony of P. aeruginosa cells resuspended in 100 uL milliQ water | ||
| + | |||
program: touchdown PCR (called U on Eppendorf) | program: touchdown PCR (called U on Eppendorf) | ||
Revision as of 12:18, 14 August 2013
14 August 2013
Contents |
lab 208
Main purpose
- PCR for extraction of Nir2 from PAO1
Who was in the lab
Gosia, Julia, Henrike
Procedure
PCR for Nir2 extraction from PAO1
We tried to reproduce the successive 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 U 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 | - |
Gel purification
gel purify PCR products from constitutive promoter SPL and constitutive promoter ref
Results
Conclusion
Navigate to the Previous or the Next Entry
