Cloning Multiplex-PCR Products into pUC Vectors for DNA Sequencing
Latest update 2-21-00
  1. PCR Product Clean-up:

    1. Add the following to each reaction:
      1. 10 units of USB shrimp alkaline phosphatase (SAP)
      2. 100 units of USB exonuclease I
    2. Incubate for 30 minutes at 37 degrees C, 10 minutes at 80 degrees C
    3. Store at 4 degrees C until further analysis
    4. Check PCR products on 1% agarose gel containing ethidium bromide (reference Roe Lab protocol book) loading 5-10 ml of the reaction products
    5. Extract the remaining sample once with an equal volume of phenol/chloroform (1:1)
    6. Precipitated with 2.5 volumes of ethanol/acetate at -20 degrees C overnight
    7. Wash with 70% ethanol
    8. Dry and resuspend in 30 ml of sterile distilled deionized water.

  2. End-repair and phosphorylation ( directly from Roe protocol book)

    1. Add the following to each reaction:

      		* 10X Kinase buffer			 5 ml
      		* 10 mM rATP				     5 ml
      		* 0.25 mM dNTPs				 7 ml
      		* T4 polynucleotide kinase	 1 ml (3 U/ml)
      		* Klenow DNA polymerase		2 ml (5 U/ml)
      
    2. Incubate at 37 degrees C for 30 minutes.
    3. Phenol/chloroform extract as above.
    4. Precipitate with ethanol-acetate as above
    5. Wash with 70% ethanol.
    6. Resuspend in ~15 ml sterile distilled deionized water.

  3. DNA ligation (from Roe Lab protocol book)

  4. Transformation (from Roe Lab protocol book)

  5. Subclone isolation (from Roe Lab protocol book)

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Bruce Roe, broe@ou.edu