| PCR PROTOCOL | | 点击： 作者： 来源： 时间： 2007-03-06 本站论坛 |
|  | For a 25ul rxn:
·Use 1ul of 60ng/ul or 100ng/ul DNA
·Use 1ul of each primer at 3.2pmole/ul concentration or 1.25ul of each primer at 100ng/ul concentration
·2.5ul 10x PCR Buffer w/ Mg (1.5mM)
·0.5ul 25mM MgCl2
·0.5ul dNTP
·0.125ul Taq
·18.375ul sterile water to equal a 25ul rxn (*if not making master mix, dilute Taq so that you can add 1ul of Taq and 17.5ul sterile water to equal a 25ul rxn) For a 50ul rxn: Use 2ul of 60ng/ul or 100ng/ul DNA Use 2ul of each primer at 3.2pmole/ul concentration or 2.5ul of each primer at 100ng/ul concentration 5ul 10x PCR Buffer w/ Mg 1ul 25mM MgCl2 1ul dNTP 0.25ul Taq 36.75ul sterile water to equal a 50ul rxn (*if not making a master mix, dilute Taq so that you can add 1ul of Taq and 36ul sterile water to equal a 50ul rxn) Keep the reagents on ice. Add the Taq last, and keep it in the freezer until you are ready to add it. Vortex briefly and quick spin. Cycle:
·95°C for 1-5minutes (usually 4min)
·95°C for 1min
·55°C for 1min Cycle 30 times
· 72°C for 1.5 to 2min (usually 2min)
·72°C for 10min ·4°C hold
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