Team:DTU-Denmark/Notebook/14 August 2013

From 2013.igem.org

(Difference between revisions)
(→Main purpose)
(→Procedure)
Line 14: Line 14:
<hr/>
<hr/>
-
===PCR===
+
===PCR for Nir2 extraction from PAO1===
 +
The extracted Nir2 fragment will be used as template for a screening PCR with USER primers
-
PCR reaction for amplification of Nir2 extraction fragment in order to obtain a huge amount to use as template to make a screening PCR for the Nir2 USER fragment
 
-
primers:
+
primers: 42a and 42b
-
template: P. aeruginosa cells resuspended in 100 uL
+
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)
 +
 +
Reaction Mix
 +
{| class="wikitable" style="text-align: right"
 +
! component(per reaction) !! without additives !! using 5% DMSO !! using 1M Betaine
 +
|-
 +
| dNTPs || 1uL || 1uL || 1uL
 +
|-
 +
| HF buffer || 10uL || 10uL || 10uL
 +
|-
 +
| X7 polymerase || 0.5uL || 0.5uL || 0.5uL
 +
|-
 +
| MilliQ water || 31.5uL || 29uL || 21.5uL
 +
|-
 +
| template || 1uL || 1uL || 1uL
 +
|-
 +
| FW primer || 3uL || 3uL || 3uL
 +
|-
 +
| RV primer || 3uL || 3uL || 3uL
 +
|-
 +
| DMSO || - || 2.5uL || -
 +
|-
 +
| Betaine || - || - || 10uL
 +
|-
 +
|}
 +
===Gel purification===
===Gel purification===

Revision as of 11:47, 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

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)

Reaction Mix

component(per reaction) without additives using 5% DMSO using 1M Betaine
dNTPs 1uL 1uL 1uL
HF buffer 10uL 10uL 10uL
X7 polymerase 0.5uL 0.5uL 0.5uL
MilliQ water 31.5uL 29uL 21.5uL
template 1uL 1uL 1uL
FW primer 3uL 3uL 3uL
RV primer 3uL 3uL 3uL
DMSO - 2.5uL -
Betaine - - 10uL


Gel purification

gel purify PCR products from constitutive promoter SPL and constitutive promoter ref

Results

Conclusion

Navigate to the Previous or the Next Entry

Retrieved from "http://2013.igem.org/Team:DTU-Denmark/Notebook/14_August_2013"