Kidd Lab 

SacI site (7.6 kb upstream of HOXB6; Clone pH 2-13-2 and pBS3)

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

Primer 1.     BS3SAC1:     5'-GAG GGG GGA GTC TCC CTC A-3'          100 ng/reaction
Primer 2.     BS3SAC2:     5'-CTG CAG GGA GAA GCT TCC CA-3'        100 ng/reaction

dNTPs: Final Concentration 200 µM each

Buffer: Final Concentration 2.25 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:  94^oC (5')
                               94^oC (30"), 61^oC (30"), 72^oC (1')  × 30 cycles

Product size:
                                     SacI
                    ~560 bp ----------------> 450 bp +  110 bp (in homozygote site present)

[NB: There is preferential amplification of the smaller product.  This can be minimized by limiting genomic DNA to 100 ng/reaction volume of 25 µl.]

Reference:
Murphy, P.D., A.C. Ferguson-Smith, T. Miki, A.A. Feinberg, F.H. Ruddle, and K.K. Kidd, 1987. A moderately frequent RFLP identified by both SacI and BanII with a probe from the HOX2 locus in man (17q11-17q22). Nucleic Acids Research 15:6311-6312.
 
 

Kidd Lab Histograms for Allele Frequencies