PCR Protocol, PCR Steps

PCR Protocol

In a traditional PCR protocol, reaction components are assembled as described below. The final volume should be 50 µL.

• Thaw all reagents on ice.
• Assemble reaction mix into 50 µL volume in a thin walled 0.2 mL PCR tubes.
• Add reagents in following order: water, buffer, dNTPs, Mg CL2, template primers, Taq polymerase.
• Gently mix by tapping tube. Briefly centrifuge to settle tube contents.
• Prepare negative control reaction without template DNA.
• Prepare positive control reaction with template of known size and appropriate primers.

Component	Final Concentration/amount
water	to 50 µL
buffer	1 x
Taq polymerase	0.05 units/µL
dNTP mix	200 µM
MgCl2	0.1-0.5 mM
Forward primer	0.1-0.5 µM
Reverse primer	0.1-0.5 µM
template	200 pg/µL
DMSO (optional)	1 to 10%  w/v

PCR Steps

• Program your thermocycler for your PCR reaction using the following guidelines:
  

  Step	Temp	Time	# of cycles
  Initial Denaturation	94°C	5 min
  Denaturation	94°C	30 sec	30-35
  Primer Annealing	Tm-5°C	45 sec
  Extension	72°C	1 min per kb
  Final Extension	72°C	5 min

• Analyze the results of your PCR reaction via gel electrophoresis.

PCR Design Tools

• PCR Primer Design
• Oligo Calculator for Tm, MV, and µg/OD
• Restriction Enzyme Map Analysis
• Common Restriction Enzyme Sites