Team:SCU-China/PCR

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Revision as of 18:53, 17 October 2014

PCR Protocol

For constructing the Biobricks that we want to submit, we set up the PCR protocol and PCR system as bellows:

PCR system (For 50μL)

Materials

Volume

Taq DNA polymerase

0.5μL

Template

0.5μL

Forward Primer

1μL

Reverse Primer

1μL

dNTPs (2.5mM)

4μL

10x Buffer (Mg2+ free)

5μL

MgCl2 (25mM)

3μL

Sterilized diluted water

35μL

Note: The content of template should be less than 500ng for the 50μL system. For all of our experiments, the content of template of 0.5μL DNA lower than 500ng.

PCR protocol

Temperature

Times

95℃

5min.

30-35 Times

95℃

30sec.

55-65℃

30sec.

72℃

1-2min.

72℃

10min.

Note: for finding the optimal annealing temperature, we have done a series of gradient of annealing temperature first. And as for the elongation time, Taq DNA polymerase could synthesize DNA as the speed of 1000-2000 nucleotides per minute.

Sichuan university

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