Team:Peking S/lab/protocol/pcr

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Protocol

Double Digestion |Gel Extraction|Miniprep | Chemical Inducible Expression of GFP |DNA Purification |Ligation of Insert DNA | PCR |Preparation of Competent Cells| Site-directed Mutagenesis| Transformation|

Protocol for PCR with EasyPfu DNA Polymerase

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Preparation for Reaction Mixture

Reagent	Final concentration	Volume
10X Taq buffer	1X	2 µl
2.5 mM dNTP mix	0.25 mM	2 µl
Forward Primer	0.2-0.4 µM	1 µl
Reverse Primer	0.2-0.4 µM	1 µl
EasyPfu DNA Polymerase	25 units	1 µl
Template DNA	As required	<0.5 µg
ddH2O	to final volume

Cycling Conditions

Step	Temperature/℃	Time/min
Initial Denaturation	94	5
Denaturation	94	0.5
Primer Annealing	As required	As required
Extending	72	As required(0.5kb/min)
Final Extending	72	10
Number of Cycles		32-35
Cooling Down	4	10

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