Kidd Lab 

EcoRI site (located in intron 5)

Template: Genomic DNA 100-200 ng/reaction volume of 25 µl

Primer 1.     PAHRIF:     5'-AGA GTT TTT AGC ATG AAA GGC-3'             100 ng/reaction
Primer 2.     PAHRIR:     5'-CTA CCC ACC AAA AAA AGT ACA-3'            100 ng/reaction

dNTPs: Final Concentration 200 µM each

Buffer: Final Concentration 2.5 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 (15"), 60^oC (15"), 72^oC (30")  × 2 cycles
                               95^oC (15"), 59^oC (15"), 72^oC (30")  × 3 cycles
                               95^oC (15"), 58^oC (15"), 72^oC (30")  × 25 cycles
                               72^oC (10')

Product size:
                                EcoRI
                   458 bp ----------------> 412  bp +  45 bp (in homozygote site present)
 
 

Kidd Lab Histograms for Allele Frequencies