Kidd Lab 

TaqI site (located in exon 13)

Template: Genomic DNA 50-100 ng/reaction volume of 25 µl

Primer 1.     RETEX13F:     5'-CTC TCT GTC TGA ACT TGG GC-3'          60 ng/reaction
Primer 2.     RETEX13R:     5'-TCA CCC TGC AGC AGG CCT TA-3'        60 ng/reaction

dNTPs: Final Concentration 333 µM each

Buffer: Final Concentration 2.25 mM MgCl₂, 50 mM KCl, 10 mM Tris-HCl pH 8.4
BSA: Final Concentration 0.16 µg/µl (e.g., 0.5 µl @ 10 mg/ml BSA/ 30 µl reaction)
DMSO: Final Concentration 5%

Taq DNA Polymerase: 0.75 Units/reaction
                                 (Perkin-Elmer Amplitaq DNA polymerase 5 U/µl, Cat. No. N808-0145)

PCR Cycling Profile: 95^oC (5')
                              95^oC (30"), 56^oC (30"), 72^oC (1')  × 30 cycles

Product size:
                                 TaqI
                   239 bp -------------->  133 bp +  106 bp (in homozygote site present)

Reference:
Romeo, G., P. Ronchetto, Y. Luo, V. Barone, M. Seri, I. Ceccherini, B. Pasini, R. Bocciardi, M. Lerone, H. Kaariainen, et al., 1994. Point mutations affecting the tyrosine kinase domain of the RET proto-oncogene in Hirschsprung’s disease. Nature367:377-378.
 
 

Kidd Lab Histograms for Allele Frequencies