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Types of PCR

• AFLP PCR
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PCR

Nested PCR Protocol Conditions

 

For 50 ul reactions, the amounts given are per reaction. Make sure you make an extra reaction for every 6 reactions to have enough master mix.

External Round of PCR

Master Mix

dNTP mix (2mM each dNTP)	5 µl
10x PCR buffer (15mM MgCl2)	5 µl
Taq polymerase (0.2U)	0.2 µl
dH2O	25.8 µl
Primer mix. Forward  and Reverse primers. (5 pmoles/µl each)	4 µl
Total Mastermix Volume:	40 µl

DNA Template

10 µl

Internal Round of PCR

Nested PCR Master Mix

dNTP mix (2mM each dNTP)	5 µl
10x PCR buffer (15mM MgCl2)	5 µl
Taq polymerase (0.2U)	0.2 µl
dH2O	35.8 µl
Primer mix. Forward  and Reverse primers. (5 pmoles/µl each)	4 µl
Total Mastermix Volume:	49 µl

DNA Template (Sample from external round PCR product)

1 µl