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