Saturday, May 15, 2004

Description
RET TaqI Polymorphism

Procedure
Taq I polymorphism located in Exon 13

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

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 in the reaction 333 uM each

Buffer: Final Concentration 2.25 mM MgCl2, 50 mM KCl, 10 mM Tris HCl pH 8.4
BSA: Final Concentration 16 ug/ul (e.g., .5 l @ 10 mg/ml BSA/30 ul Rx)
DMSO: Final Concentration 5%

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

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

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

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. Nature 367:377-378.


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