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Team:EPF-Lausanne/Protocols/T7-ext
From 2011.igem.org
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* 0.5 ul 3' ext primer (500 nM) | * 0.5 ul 3' ext primer (500 nM) | ||
* 1 uL product of gene specific PCR | * 1 uL product of gene specific PCR | ||
| - | 0.5 ul Hifi+ | + | * 0.5 ul Hifi+ |
* to 50 ul H2O | * to 50 ul H2O | ||
10 cycles | 10 cycles | ||
| - | annealing temperature = 55°C | + | |
| + | annealing temperature = 55°C | ||
| + | |||
extension time = depends on length (about 1kb/minute) | extension time = depends on length (about 1kb/minute) | ||
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When the previous PCR is done, open the PCR machine (don't wait too long) and add 0.5 uL of each final | 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) (I made a mix, so just add 1 uL of it). 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}} | ||
Latest revision as of 14:04, 18 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 = depends on length (about 1kb/minute)
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) (I made a mix, so just add 1 uL of it). Use the same protocol as before but run for 35 cycles and set annealing temperature as 47°C (or 50°C).
