Polymerase Chain Reaction (PCR) 5 ul 10X PCR buffer (contains buffering agent, KCl, 15mM MgCl2) 4 ul dNTPs, stock is 2.5 mM (each nucleotide is at 2.5 mM) 2 ul 5’ primer, 10 pmol/ul stock 2 ul 3’ primer, 10 pmol/ul stock water (Millipore filtered), q.s. to 50 ul (quantity sufficient to give 50 ul) 1.0 ul taq polymerase 1-5 ul DNA template (about 100-500 ng) 50 ul total volume a. make a cocktail for the number of reactions plus 1 extra. Make sure to include a neg. control reaction that lacks template. Dole out cocktail mixture and then add template. b. can cut recipe in half (25 ul total volume reactions) c. alternatively, use a 2X Taq polymerase mixture that contains polymerase, buffer, salts and dNTPs; just add primers, template and water. d. When screening bacterial colonies for recombinant plasmids, pick a portion of a colony using a pipet tip or sterile toothpick and swirl into reaction in place of template. Include a neg. control colony reaction. -Typical PCR conditions: 1. 94 C for 5 minutes 2. 94 C for 30 seconds 3. 55 C for 30 seconds (temp. depends on Tm of primers; want ~5° below lowest primer Tm) 4. 72 C for 1’ (1 minute per kb of target DNA) 5. -repeat back to step 2, 29-39 times depending on target template conc. (34 is typical). 6. 72 C for 10 minutes to finish DNA ends 7. hold at 4 C (so you can go home without worry) 8. store at -20 C until further processing 9. add Loading buffer to 1-2X. Gel check 5-10 ul. ****************************************