Direct Sequencing from Amplified Bacterial and Large Insert Cloned Human or Mouse Genomic DNA via an Improved MultiPlex PCR-based Method
Latest update 7-20-00
  1. Preparing primers for MP-PCR

      The average concentration of a Mermade-synthesized primer is ~19 pmole/ul. To prepare the primers for MP-PCR, one ul of each primer is pooled into a microfuge tube and dryed. The dryed primers are then suspended in 10 ul sterile distilled deionized water. You might not need to this if you have few primers and just simply pool them into the reaction tube directly.

  2. Amplification:

    1. In a 96-well thermocycle plate (or each of four 0.5 ml microfuge tubes), the following was added: 
      	10 ul of 5X PCR buffer 
		(83 mM (NH4)2SO4, 335 mM Tris-HCl (pH 9.0),  33.5 mM MgCl2, 50 
		mM b-mercaptoethanol, and 850 ug/ml bovine serum albumin)  
 	10 ul of 96 concentrated primers (~20 pmole each)
	~250 ng genomic or large insert BAC, PAC, Fosmid or Cosmid target template DNA
	12 ul of dNTP mix (25 mM each)
	2 units of Taq polymerase XL (Perkin-Elmer)
	2.5 ul DMSO
	Sterile distilled deionized water to bring the final reaction volume to 50 ul.
    2. Thermocycle (Perkin-Elmer Cetus 9600 or 4800) conditions: 
                      *6 minutes at 94 degrees C  
 		*30-40 cycles of: 
			denaturation for 30 seconds at 94 degrees C
			annealing for 30 seconds at 55 degrees C
			extension for 4 minutes at 65 degrees C 
		*4 degrees C until further analysis.
  3. PCR Product Clean-up:

      Depending on the number of primers used, you can use one of following clean-up protocols:

    1. For more than 20 pooled primers:

      1. Add the following to each reaction: * 10 units of shrimp alkaline phosphatase (SAP) * 100 units of 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 ul of the reaction products
      5. Extract the remaining sample once with an equal volume of phenol/chloroform (1:1)
      6. Precipitate with 2.5 volumes of ethanol/acetate
      7. Wash with 70% ethanol
      8. Dry and resuspend in 50 ul of sterile distilled deionized water.
    2. For less than 20 pooled primers:

      1. Precipitate with 2.5 volumes of absolute 100% ethanol.
      2. Wash with 70% ethanol.
      3. Dry and resuspend in 50 ul of sterile distilled deionized water
      4. Filter through Sephadex G50 (reference Roe Lab protocol book)

  4. Sequencing:

    1. Distribute 2 ul of the PCR products to each well of a Robbins 96 well cycle sequencing microtiter tube plate (depending on the number of primers used to generate the PCR products).
    2. Add 2 ul of each primer used in the PCR reaction individually to each well
    3. Add 1 ul 25% DMSO and 1 ul of BigDye mix to each well
    4. Thermocycle according to the BigDye thermocycling conditions: 
      		5 minutes at 95 degrees C once
		60 cycles of 
		     30 seconds at 95 degrees C
		     20 seconds at 50 degrees C
		     4 minutes at 60 degrees C
		Hold at 4 degrees C
    5. Filter through Sephadex columns in 96 well microtiter plate format (refer to the Roe Lab protocol book)

This protocol was adapted from:
J.S. Chamberlan, R.A. Gibbs, J.E. Ranier and C. T. Caskey, "Multiplex PCR for the Diagnosis of Duchenne Muscular Dystrophy" in PCR Protocols, eds. M. A. Innis, D.H. Gelfand, J.J. Sninsky and T. J. White (Academic Press, NY, 1990) pp. 272-281

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Bruce Roe, broe@ou.edu