Overlap extension PCR

Overlap extension PCR

This type of PCR is used to make mutations, fuse two gene segments together, make insertions within a gene, or make deletions within a gene.

Set up the two half reactions as follows:

Vent Polymerase Reactions for the two halves:

• 80.5 ul distilled H₂O
• 10 ul ThermoPol buffer
• 2 ul 10 mM dNTPs
• 2 ul 100 mM MgSO₄
• 2 pmoles primerA or B, usually 2 ul of a 100 pM stock
• 2 pmoles primerC or D, usually 2 ul of a 100 pM stock
• 1 ul miniprep or Qiagen DNA, usually around 200-500 ng template
• 0.5 ml Vent DNA Polymerase

Note: Volume of distilled H₂O can be adjusted if additional volumes of reagents are required.

PCR Cycle (for PE 9600 or equivalent):..

Read Full Protocol here

http://www.gardnerlab.org/protocols/oePCR.htm

Gardner Lab Research: Overlap Extension PCR