Kidd Lab 

PstI (A) site (located between HindIII(A) and TaqI sites)

Template: Genomic DNA 100-200 ng/reaction volume of 20 µl

Primer 1.     PKO82OUT2:      5'-GTA CAG GAT TCA CAG GAC TC-3'          100 ng/reaction
Primer 2.     82OUT3CK3:       5'-CCT TCC CCT TGA ATC TGA GCC-3'         100 ng/reaction

dNTPs: Final Concentration 200 µM/reaction

Buffer: Final Concentration 1.5 mM MgCl₂, 50 mM KCl, 10 mM Tris-HCl pH 8.4

Taq DNA Polymerase: 0.5 Units/reaction
                                 (Perkin-Elmer Amplitaq DNA polymerase 5 U/µl, Cat. No. N808-0145)

PCR Cycling Profile: 95^oC (5')
                              95^oC (30"), 62^oC (30"), 72^oC (30")  × 30 cycles

Product size:
                                 PstI
                    700 bp--------------> 400 bp + 300 bp (in homozygote site present)

Reference: PstI RFLP site located in Kidd lab by sequencing.( Note: This amplicon also contains the polymorphic BglI site. The two alleles are GCN₅GGC and GCCN₅AGC giving BglI digestion fregments of 606 and 113 bp when the site is present)
 
 

Kidd Lab Histograms for Allele Frequencies