BioProtocols--pcr product cleanup

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PCR Product Cleanup

PCR Cleanup for Sequencing

To remove excess dNTPs and unincorporated primers, use the Exo-SAP method below. This is usually NOT necessary for cleaning up amplicons prior to sequencing. Simply diluting the PCR product ten or twenty fold will suffice to reduce any interference leftover primers or dNTPs would cause in the sequencing reaction.

Materials

PCR product
Exo-SAP (Amersham Biosciences, Cat # US78200, 60 cents to 95 cents per reaction, or use the recipe below)

a) 78 ul ddH20

b) 2 ul Exonuclease I (Amersham Biosciences cat# E70073X, 10 U/ul, 4.02 cents per unit, or cat# E70073Z at 4.4 cents per unit)

c) 20 ul Shrimp Alkaline Phosphatase (Amersham Biosciences cat# E70092X, 1 U/ul, 7.62 cents per unit, or cat# E70092Z at 11.9 cents per unit)

d) 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 the 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 37C for 20 min (degrade primers and dephosphorylate dNTPs)
Incubate at 80C for 15 min (destroys the enzymes)