Team:DTU-Denmark/Notebook/14 August 2013

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===PCR for Nir2 extraction from PAO1===
===PCR for Nir2 extraction from PAO1===
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We tried to reproduce the successive extraction of Nir2 from PAO1.
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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.
The extracted Nir2 fragment will be used as template for a screening PCR with USER primers.
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===PCR for Nir2 with USER endings===
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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.
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Sample 1-6: 63C annealing temperature
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Sample 7-12: ramp from 63C-57C
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If this PCR is not successful we suggest to try touchdown.
===Gel purification===
===Gel purification===

Revision as of 12:59, 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 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 63C-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

Conclusion

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