• Introduction
  • Home
  • The Team
  • Photo Gallery
• Our Project
  • Results Summary
  • Selection System
  • In-Vitro Characterization
  • In-Vivo Characterization
  • Microfluidics
  • Data
  • Attributions
• Tools
  • Gibson Assembly
  • MITOMI
• Notebook
  • Protocols
  • May
  • June
  • July
  • August
  • September
  • October
• Considerations
  • Human Practices
  • Safety
• Acknowledgements
  • Sponsors
  • Partners

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). Use the same protocol as before but run for 35 cycles and set annealing temperature as 47°C (or 50°C).