Kidd Lab 

1. Polymerase Chain Reaction (PCR)

Template: Genomic DNA 100-200 ng/reaction volume of 10 µl

Primer 1.     TDI_EX12.1:     5'-CGA GTA CGG GCT GCA GGC ATA CAC T -3'            100 ng/reaction
Primer 2.     EX12R4 :         5'-CCC AGC AGG TCC CAC AaT CAC AG-3'                    100 ng/reaction

dNTPs: Final Concentration 170 µM each

Buffer: Final Concentration 2.5 mM MgCl₂, 50 mM KCl, 15 mM Tris-HCl pH 8.0

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

PCR Cycling Profile: 94^oC (10')
                              94^oC (15"), 58^oC (15"), 72^oC (15")  × 25 cycles
                              72^oC (10')

Product size:
                    58 bp
 
 

2. Fluorescence Polarization method

2(1). Primer and dNTP degradation

Buffer: Final Concentration 5 mM MgCl₂, 10 mM Tris-HCl pH 8.0

Alkaline phosphatase: Final concentration 14U/reaction

ExonucleaseI: Final concentration 1U/reaction

Incubation: 37°C (90')

2(2). SNP genotyping Assay

Buffer: Final Concentration 6.5 mM MgCl₂, 26 mM Tris-HCl pH 9.5

Internal detection primer: Final concentration 8pmol

Dye-labeled ddNTP mix: Final concentration 2pmol

ThermoSequenase: Final concentration 0.64U/reaction

Single Base Extension Reaction: 94°C (1')
                                                 94^oC (10"), 55^oC (30"),   × 25 cycles
 
 

Kidd Lab Histograms for Allele Frequencies