Kidd Lab 

Primer 1.     RET19F:   5'-GGC AAA CAG CGG GGA AGC ACA-3'
Primer 2.     RET19R:   5'-CCA CTG ACG ATG AGG CAA GAA-3'

Concentrations per reaction (final):

            Mg⁺⁺ concentration         1.5mM
            Primer amount                10ng
            dNTP concentration       0.02mM
            Amplitaq GOLD               0.5units
            DNA                                   100 ng

PCR Cycling Profile:

             94^oC (10')
             94^oC (30"), 56^oC (30"), 72^oC (1')   × 30 cycles
             72^oC (7')

2. SAP/EXO clean-up reaction:

Per reaction (final concentration):

            Buffer (USB 70103)                                    .25X
            Shrimp Alkaline Phosphatase                 2Units
            Exonuclease                                                0.1unit

Cycling Profile:

             37^oC (90')
             95^oC (30')

3. Single base extension:

Primer used (reverse primer): 5’-TCA GCA GCA GGA AGG AGC AGA AA-3’

Per reaction (final concentration):

            Buffer ( USB 78500 )                                    .2X
            ddNTPs                                                          25nM
            ( R110 ddCTP. TAMRA ddGTP, unlabelled ddATP and dd UTP)
            primer amount                                              8ng
            Thermosequenase                                      0.2Units

Cycling Profile:

             93^oC (2')
             93^oC (10"), 55^oC (30")  × 20 cycles

Reference:
Chen et al., 1999 May.  Fluorescence polarization in homogeneous nucleic acid analysis.  Genome Res. 9(5): 492-8.
 
 

Kidd Lab Histograms for Allele Frequencies