Denise Yu: Difference between revisions
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Reagent | Reagent | ||
-10x Thermo-Start Standard Buffer (5 microliters) | -10x Thermo-Start Standard Buffer (5 microliters) | ||
-20mM dNTP Mix (2 microliters) | -20mM dNTP Mix (2 microliters) | ||
-25mM MgCl2 (3 microliters) | -25mM MgCl2 (3 microliters) | ||
-Primer Mix (2.5 microliters) | -Primer Mix (2.5 microliters) | ||
-Template DNA 10 ng | -Template DNA 10 ng | ||
-Thermo-Start DNA Polymerase (.25 microliters) | -Thermo-Start DNA Polymerase (.25 microliters) | ||
-double-distlled, sterile H2O (up to 50 microliters) | -double-distlled, sterile H2O (up to 50 microliters) | ||
Thermal Cycle | Thermal Cycle | ||
Enzyme Activation | Enzyme Activation | ||
1. 95C for 15 minutes | 1. 95C for 15 minutes | ||
Denaturation | Denaturation | ||
2. 92-95C for 10-30 seconds | 2. 92-95C for 10-30 seconds | ||
Annealing | Annealing | ||
3.50-65C for 30 seconds, repeart 25-30 | 3.50-65C for 30 seconds, repeart 25-30 | ||
Extension: | Extension: | ||
4.72C for 30 seconds | 4.72C for 30 seconds | ||
Final Extension: | Final Extension: | ||
5. 72C for 5 minutes | 5. 72C for 5 minutes |
Revision as of 23:47, 10 November 2008
Hot Start PCR
Reagent
-10x Thermo-Start Standard Buffer (5 microliters)
-20mM dNTP Mix (2 microliters)
-25mM MgCl2 (3 microliters)
-Primer Mix (2.5 microliters)
-Template DNA 10 ng
-Thermo-Start DNA Polymerase (.25 microliters)
-double-distlled, sterile H2O (up to 50 microliters)
Thermal Cycle
Enzyme Activation
1. 95C for 15 minutes
Denaturation
2. 92-95C for 10-30 seconds
Annealing
3.50-65C for 30 seconds, repeart 25-30
Extension:
4.72C for 30 seconds
Final Extension:
5. 72C for 5 minutes