PCR Genotyping of Mouse Tails for IRS-1 and IRS-2

PCR Genotyping of Mouse Tails for IRS-1 and IRS-2

Make up stock solution of digestion buffer, leaving out b-ME.

Digestion Buffer

components	for 100 ml
6.7 mM Tris-HCl, pH 8.8	3.35 ml of 2 M stocks
1.66 mM (NH₄)₂SO₄	1.66 ml of 1 M stocks
0.67 mM MgCl₂	1.34 ml of 0.5 M stocks
0.5% Triton X-100	0.5 ml Triton X-100
Autoclaved H₂O	92.15 ml of ddH₂O (autoclaved)

1% b-mercaptoethanol	1.0 ml b-mercaptoethanol

For “X” tails, place [(99ml)(“X”+6)] digestion buffer in well of 12- or 6-well plate (label well for re-use). Add (“X”+6)ml b-ME to well and swirl to mix. Use repeat-pipettor (set to “1” with correctly-labeled “PCR Digestion Buffer” eppendorf combi-tip (5ml capacity)) to add 100ml digestion buffer to a properly labeled microfuge tube containing a small piece of mouse tail (approximately 1/16-inch).

Cap tubes and place in hot block (room 630, balance area) for 10 minutes (check that thermometer is set to at least 95ºC).

Using repeat-pipettor (with 500ml-combi-tip labeled “proteinase K”), set to “1”, add 10ml of 10mg/ml stocks of proteinase K (found in –20 in labeled bottle – some are –20mg/ml but not labeled) to each microfuge tubes. Cap and place in plastic tube-box; cover box and secure with rubber band. Place box in 55ºC water bath, room 635 on Liangyou’s bench for at least 2 hours. Tails can sit in this bath overnight.

Remove box from bath and remove tubes to styrofoam tube-holders. Place tubes in hot block for another 10 minutes at 95ºC to heat-denature the proteinase K. Spin tubes on high for 5 minutes. Place in white boxes, label, and place in cold room for storage.

PCR set-up

Add 1ml tail DNA (from top of tube contents – don’t take fur debris at bottom) to PCR tubes – keep record of which DNA samples are in which tubes.

Make up PCR mix for appropriate gene(s), as follows:

To assess IRS-1 endogenous band, use IRS-1 upper and IRS-1 lower primers.

To assess IRS-1 neo band, use IRS-1 lower and IRS neo primers.

To assess IRS-1 complete double-band reaction, use IRS-1 upper, lower, and IRS neo primers.

To assess IRS-2 endogenous band, use IRS-2 upper and IRS-2 lower primers.

To assess IRS-2 neo band, use IRS-2 lower and IRS neo primers.

To assess IRS-2 complete double-band reaction, use IRS-2 upper, lower, and IRS neo primers.

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

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

IRS neo: gct acc cgt gat att gct gaa gag

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

IRS-2 lower: agc tct gga ggt tta ctt tcc tag

PCR Mix Ingredients for single-band reaction

18.5 ml water

2.5 ml RedTaq 10X buffer

0.5 ml primer 1 (previously diluted to 10pm/ml)

0.5 ml primer 2 (previously diluted to 10pm/ml)

1.0 ml dNTPs (previously diluted to 4 mM)

1.0 ml RedTaq polymerase (5 U/ml)

PCR Mix Ingredients for double-band reaction

17.5 ml water

2.5 ml RedTaq 10X buffer

0.5 ml primer 1 (previously diluted to 10pm/ml)

1.0 ml primer 2 (previously diluted to 10 pm/ml)

0.5 ml primer 3 (previously diluted to 10pm/ml)

1.0 ml dNTPs (previously diluted to 4 mM)

1.0 ml RedTaq polymerase (5 U/ml)

… where “primer 2” is always either IRS-1 lower or IRS-2 lower, to ensure that there is enough of these two primers to anneal with both the upper and neo primers.

PCR Profile for IRS-1 and IRS-2

94ºC 1 minute

94ºC 30 seconds

60ºC 30 seconds 30 cycles

72ºC 1 minute, 30 seconds

72ºC 10 minutes

4ºC forever

Use the above profile for all PCR reactions for IRS-1 and IRS-2. This is a different profile from the one that worked originally with these primers, one that included an annealing temperature of 65ºC. Beginning in October, 2000, this previous protocol stopped working, despite the fact that the Tms listed on the primers allowed for such an annealing temperature. We lowered the annealing temperature to 60ºC and everything worked. When in doubt, change the annealing temperature.

Product Sizes

IRS-1: 360WT, 680 Rec

IRS-2: 530 WT, 650 Rec