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DNA Sequencing Facility
* PCR product
* Exo-SAP (Amersham Biosciences, Cat # US78200, or use the recipe below)
78 ul ddH20
2 ul Exonuclease I (Amersham Biosciences cat# E70073X, 10 U/ul, or cat# E70073Z)
20 ul Shrimp Alkaline Phosphatase (Amersham Biosciences cat# E70092X, 1 U/ul, or cat# E70092Z)
100 ul Total
1. Perform PCR amplification.
2. Check the product on an agarose gel with a size standard.
3. 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.
4. If a single band has been obtained, then mix 10 ul PCR product with 4 ul Exo-Sap.
5. Incubate at 37C for 20 min (degrade primers and dephosphorylate dNTPs)
6. Incubate at 80oC for 15 min (destroys the enzymes)
Unless otherwise specified, all data and reagents distributed by the University of Wisconsin Biotechnology Center DNA Sequencing Facility are intended for research purposes only. They are not intended nor certified for diagnostic or clinical use. Clinical services are provided through our collaboration with the UW Collaborative Genomics Core.