Enzymatic PCR purification
After PCR amplification your samples contain leftover deoxynucleotides and primers which were not consumed during amplification. Extra nucleotides and primers has to be removed before sequencing reaction or they will severely affect the quality of the results. (see picture).
PCR purification can be done e.g enzymatically, by filtering or by gel purification. Two simple protocols are presented below for enzymatic purification.
A) ExoI - SAP protocol
PCR products are treated with Exonuclease I (ExoI) and Shrimp Alkaline Phosphtase (SAP).
- Exonuclease I degrades ssDNA (excess PCR primers) from the sample.
- SAP dephosphorylates deoxynucleotides thus preventing them to participate in sequencing reaction.
ExoI and SAP enzymes are available from major laboratory suppliers.
Prepare following master mix:
- Add 5,0 µl of master mix to each sample well containing 5,0 µl of PCR product.
- Seal properly and spin down
- Incubate 60 minutes in 37ºC
- Inactive by heating to 72ºC for 15 minutes
Store in fridge until sequencing reactions
- Do not freeze - freezing may fragment your purified PCR products generating ssDNA molecules which may function as primers in your reactions.
B) ExoSAP-IT kit (USB) -download brief protocol by USB
ExoSAP-IT kit is a ready made cocktail for PCR purification.
- Add 2,0 µl ExoSAP-IT reagent in to 5 µl of sample.
- Seal plate and spin down
- Incubate 37ºC for 15 minutes
- Inactivate by heating 80ºC for 15 minutes.