Uniplex PCR-based Sequencing
 Latest update 2-21-00
  1. PCR Reactions:

      Although the PCR reaction are as described in the Roe-Lab protocol book, they are given here as well

    1. Typically, each PCR reaction contains: 
                      1 ul            target DNA (10-20 ng)
                2.5 ul        each primer (40 uM )
                1 ul            AmpliTaq DNA Polymerase (5 U)
                10 ul          2 mM dNTPs (2 mM each dNTP)
                10 ul          10X PCR buffer (500 mM KCl;
                                    100 mM Tris-HCl, pH 7.6, 10 mM
                                    MgCl2 in sterile water)
                73 ul          ddH20
                ------
                100 ul
    2. Thermocycling conditions (25 cycles): 
                      95degC for 1 minute
                55degC for 1 minute (can be reduced for less
                                    stringent hybridization)
                72degC for 2 minutes
  2. Processing PCR products:
    1. Remove any unreacted PCR primers by gel filtration of the PCR reaction by passage through sephadex G-50 (can be done on the Hydra following the protocol for DNA sequencing reaction cleanup) and then add 2.5 volumes of 100% ethanol to precipitate the PCR products.
    2. Perform the combined fillin-kinase reactions as described for shotgun cloning.
    3. Add 5 ul of agarose gel loading dye and load the reaction into a well of a low melting temperature 1% agarose gel. Electrophorese for 30-60 minutes at 100-120 mA and then excise the desired band visualized under UV light with a clean razor blade.
    4. Elute the DNA from the gel by standard freeze-thaw methods, followed by phenol extraction and concentration by EtOH-OAc precipitation.
    5. Resuspend the dried PCR products in 5-10 ul of sdd-water. Use this DNA in a standard blunt ended ligation reaction. Typically, 2-3 ul of the PCR products are combined in a 10 ul ligation reaction with 20 ng of calf intestine alkaline phosphatase treated linear double stranded pUC cloning vector (Pharmacia), although the exact amount may vary depending on the yield of amplified DNA from the PCR reaction.

  3. Alternative PCR Reaction Cleanup steps instead of passage through Sephadex G-50, usually not performed:
    1. The reaction can be cleaned using 1 ul of SAP (1 unit/ul USB # E779Y) and 1 ul of Exonuclease-I (10 units/ul USB #700073Z). This cleanup reaction mix is incubated at 30 degress for 30 minutes and then the enzymes are deactivated by heating at 80degC for 10-20 minutes.
    2. A phenol:chloroform extraction.

  4. For direct sequencing without cloning:
    1. dilute the PCR products with an equal volume of water (1:2 dilution)
    2. use 2-4 ul of the products for sequencing using the 1/8 BigDye dilution protocol.

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Bruce Roe, broe@ou.edu