I need your valuable suggestion in my Research
I have to amplify HVR-1 of malarial patients infected with Plasmodium falciparum
For which i have to perform two PCR
Primary and Nested PCR
The reaction preparation and conditions are given as follows:
Taq Buffer: 2.5 microlitre
MgCl2: 1.5 Microlitre
Taq polymerase: 1 microlitre
dNTPs: 1.5microlitre
Primers:1 microlitre Each (F& R)
DNA: 2 microlitre
while for Nested PCR i dilute the Primary PCR products 10X and then use 2 microlitre of it.
PCR water: 14.5microlitre
Conditions:
Stage 1: Primary Denaturation: 94 degree for 5 Mins
Stage 2: Denaturation: 94 degree for 1 minute
Annealing: 55 degrees for 1 minute
Extension: 72 degrees for 2 Minutes
Stage 3: Final Extension: 72 degrees for 7 Mins
No. of Cycles: 35