IRS-1 and IRS-2 Genotyping (PCR)


Digestion Buffer

      6.7 mM Tris-HCl, pH 8.8

      1.66 mM (NH4)2SO4

      0.67 mM MgCl2

      0.5% Triton X-100

      autoclaved H2O


Make up 100 ml stock.  Add 1% (final concentration) b-mercaptoethanol just prior to use.


1.       Cut very small tail piece (1/16 inch) and add 100 ml of buffer.

2.       Heat-inactivate at 95ēC for 10 minutes. 

3.       Add 5 ml of 20 mg/ml stocks of proteinase K. 

4.       Digest at 55ēC for 2 hours (can go overnight). 

5.       Heat-denature the proteinase K at 95ēC for 10 minutes. 

6.       Spin briefly (<5 min) to pellet tail debris. 

7.       Aliquot off the top. 

8.       Use 1 ml of DNA in 25 ml reaction.


Primer Sequences:


IRS-1 upper: gcc agg cac cag cat ctt cg

IRS-1 lower: tgg ccg ctc ccg aat tca at

IRS-1 neo: agc tct gga ggt tta ctt tcc tag


IRS-2 upper: ctt ggc tac cat gtt gtt att gtc

IRS-2 lower: gct acc cgt gat att gct gaa gag

IRS-2 neo: same as IRS-1 neo




1 ml DNA

17.5 ml water

2.5 ml RedTaq 10X buffer

0.5 ml upper primer        (diluted to 10pm/ml)

1.0 ml lower primer      (diluted to 10pm/ml)

0.5 ml neo primer      (diluted to 10pm/ml)

1.0 ml 4mM dNTPs

1.0   ml RedTaq


PCR Profile:


94ēC                 1 minute

94ēC                 30 seconds

65ēC                 30 seconds                                                           

72ēC                 1 minute, 30 seconds

repeat 2-4            30 cycles

72ēC                 10 minutes 


The expected band sizes are 360bp WT and 680bp rec for IRS-1; 530bp WT and 660bp rec for IRS-2.  Run on 1.5% agarose gel.