Erickson:PCR Clean-up for Sequencing

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Current revision (12:50, 27 May 2009) (view source)
 
Line 15: Line 15:
     Perform PCR amplification
     Perform PCR amplification
     Check the product on an agarose gel with a size standard
     Check the product on an agarose gel with a size standard
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     If there is more than one produce, cut out the bands separately, elute the DNA, and reamplify.  Check the product of the  
+
     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  
     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
     generated from the original larger band, clone the larger band and sequence teh insert using primers complementary to the

Current revision

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)
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