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Team:EPF-Lausanne/Protocols/T7-ext
From 2011.igem.org
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== Final PCR == | == Final PCR == | ||
| - | + | When the previous PCR is done, open the PCR machine (don't wait too long) and add 0.5 uL of each final | |
-gibson primers (at 50 uM). Use the same protocol as before but run for 35 cycles and set annealing temperature as 47°C (or 50°C). | -gibson primers (at 50 uM). Use the same protocol as before but run for 35 cycles and set annealing temperature as 47°C (or 50°C). | ||
{{:Team:EPF-Lausanne/Templates/Footer}} | {{:Team:EPF-Lausanne/Templates/Footer}} | ||
Revision as of 13:08, 15 August 2011
T7 extension PCR
Back to protocols.This protocols consists in 3 steps:
- Gene specific-PCR: amplifies the gene you want to put under the control of the T7 promoter adding RBS upstream and overhangs for the next PCR
- Extension PCR: adds the T7 promoter (or its variants) upstream the gene and overhangs for the Gibson assembly
- Final PCR: amplifies the fragments extending the gibson overhangs
Gene specific-PCR
Just a normal PCR to amplify the genes. The primers should include the RBS and overhangs with the extension primers
Extension PCR
With Hifi+ enzyme:
- 10 uL 5X Buffer
- 1 uL dNTPs
- 0.5 ul 5' ext primer (500 nM)
- 0.5 ul 3' ext primer (500 nM)
- 1 uL product of gene specific PCR
0.5 ul Hifi+
- to 50 ul H2O
10 cycles annealing temperature = 55°C extension time = 1'
Ask Henrike for the primers.
Final PCR
When the previous PCR is done, open the PCR machine (don't wait too long) and add 0.5 uL of each final -gibson primers (at 50 uM). Use the same protocol as before but run for 35 cycles and set annealing temperature as 47°C (or 50°C).
