IRS-1 and IRS-2 Genotyping (PCR)
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.