Sunday, February 01, 2004

Description
High GC content can prohibit the PCR reaction due to improper denaturing of the template or annealing of the primers. This minor spin on a common protocol is simple but works incredibly well.

Procedure
1. Basically design your PCR as you have previously with regards to annealing temp, primers, melting temp, and extension times.
2. Perform multiple reactions titering in increasing amounts of DMSO. I would recommend staring with 1% final and going up to 4% final.
3. Do not exceed 4% as that will cause the polymerase to make errors.

Recipes
1. All recipes are from whatever kit or homemade PCR reagents you have.
2. Dilute standard tissue culture DMSO to 20% and use this as a stock for the PCR reactions.

Supplies
Standard tubes will be fine for use with DMSO.
Care should be taken with DMSO as it is highly toxic.

Tips
Filter tips for the PCR advisable.