Erickson:PCR Clean-up for Sequencing
PCR Clean-up for Sequencing
To remove excess dNTPs and unincorporated primers, use the Exo-SAP method below.
Materials: PCR product Exo-SAP (recipe for 100 uL, enough to clean 25 PCR reactions) 10 uL Antartic Alkaline Phosphatase buffer (10x) 68 uL ddH20 2 uL Exonuclease I 20 uL Antarctic Alkaline Phosphatase 100 uL total
Procedure: Perform PCR amplification Check the product on an agarose gel with a size standard If there is more than one product, cut out the bands separately, elute the DNA, and reamplify. Check the product of the second amplification on a gel again to determine if multiple bands are due to duplicated primer sites. If multiple bands are generated from the original larger band, clone the larger band and sequence teh insert using primers complementary to the vector. If a single band has been obtained, then mix 10 uL PCR product with 4 uL Exo-SAP Incubate at 37 degrees C for 20 minutes (to degrade primers and dephosphorylate dNTPs) Incubate at 80 degrees C for 15 minutes (to destroy the enzymes)