Standard PCR amplification protocol used for data base primer sets.

All primer sets listed on this web site amplify unique bands under the standard conditions listed below. The same basic PCR protocol is used for both the initial primer testing (on agarose gels) and the polymorphic testing (on acrylamide gels) except that polymorphic testing uses radioactive dCTP.

Basic PCR reaction mix (10 μl)

Reagents	Amount added (1 X)	Final concentration
10X Buffer/15mM MgCl₂	1.0 μl	1 X buffer/1.5mM MgCl₂
2 mM dNTPs (dATP, dGTP, dTTP, dCTP)	1.0 μl	0.2 mM
5 units/μl Taq	0.1 μl	0.05 Units/μl
10 μM Forward primer	0.5 μl	0.5 μM
10 μM Reverse Primer	0.5 μl	0.5 μM
10ng/μl DNA	1.0 μl	1.0 ng/μl
PCR compatible Dye^*	2.0 μl
dH₂O	3.9 μl
Total	10.0 μl

PCR amplification protocol

Step	Temperature	Time	Cycles
Initial Denaturing	94⁰C	4 min	1
Denaturing	94⁰C	1 min
Annealing	58⁰C	1 min	] 30
Elongation	72⁰C	1 min
Final elongation	72⁰C	5 min	1

^*PCR compatible Dye contains 12% sucrose, 0.8 mg/ml tartrazine, 0.1mg/ml cresol red.
Filter and aliquot in 1.5 ml tubes and store at -20⁰C. Adding PCR compatible dye
saves time by allowing the mix to be loaded directly onto a gel.