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Team:EPF-Lausanne/Protocols/T7-ext
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This protocols consists in 3 steps: | This protocols consists in 3 steps: | ||
| - | * Gene | + | * 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 | * 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 | * Final PCR: amplifies the fragments extending the gibson overhangs | ||
| Line 13: | Line 13: | ||
== Extension PCR == | == 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). | ||
{{: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).
