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T7 extension PCR

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).