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

ReagentFinal concentrationVolume
10X Taq buffer1X2 µl
2.5 mM dNTP mix0.25 mM2 µl
Forward Primer0.2-0.4 µM1 µl
Reverse Primer0.2-0.4 µM1 µl
EasyPfu DNA Polymerase25 units1 µl
Template DNAAs required<0.5 µg
ddH2Oto final volume

Cycling Conditions

StepTemperature/℃Time/min
Initial Denaturation945
Denaturation940.5
Primer AnnealingAs requiredAs required
Extending72As required(0.5kb/min)
Final Extending7210
Number of Cycles 32-35
Cooling Down410