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.
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.
*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.
Depending on the number of primers used, you can use one of following clean-up protocols:
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
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
Bruce Roe, email@example.com