Base Deletion by Site-Directed Mutagenesis with Extreme GC Content

Project Description

Small 1 base deletion from an Extreme GC Content template (up to 94% GC-rich)

This project originated from previous exchanges with a customer having problems with Extreme GC Content PCR amplification. Their plasmid codes for a sfGFP-GOI in pcDNA3. The researcher’s lab is located at the the Université de Montréal in Montreal. The problem is that their GOI has Extreme GC Content. The first 200 bp of hHCN2 has a minimal GC content of 92%.Then, the GC content does not drop below 76% for at least another 400 bp. Overall, the 3006 bp coding for the GOI has a GC content of 70%. Total vector size is 9108 bp. That sure sounded like a nice challenge for us to solve!

Mutagenesis PCR reaction setup:

• H2O : complete to 25 ul
• 5x buffer: 5 ul
• dNTPs (2.5 mM): 2 ul
• Forward primer (10 uM): 0.5 ul (200 nM final)
• Reverse primer (10 uM) : 0.5 ul (200 nM final)
• sfGFP-hHCN2/pcDNA3 plasmid : 1 ul (10 ng)
• PCR Stimulant: 0, 5 or 10 ul
• Polymerase: 0.5 ul

PCR cycling for site-directed mutagenesis :

1. Denaturation: 120s at 94 °C
2. 25 x
   1. Denaturation: 30s at 94 °C
   2. Annealing: 20s at 58 °C
   3. Extension: 500s at 72 °C
3. Final extension: 600s at 72 °C