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

PCR Genotyping of Mouse Tails for IRS-4

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-4 endogenous band, use IRS-4 cre c and IRS-4 cre d primers.

To assess IRS-4 neo band, use IRS-4 cre a and IRS-4 cre c primers.

To assess IRS-4 complete double-band reaction, use IRS-4 cre a, cre c, and cre d primers.

IRS-4 cre a: ggt tgc act tac acc tta cac tcc

IRS-4 cre c: ggg cta cac ctg cgc taa atg gg

IRS-4 cre d: aag taa tgt ttg ctg atg ata ccc

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 IRS-4 cre c, to ensure that there is enough of this primer to anneal with both the cre a and cre d primers. The double-band reaction has yet to work – perhaps bands are too close together or competition for cre c band is too great to see both bands. Use single-band reactions, particularly because you only need to know the endogenous band for girls (IRS-4 is on the X chromosome).

PCR Profile for IRS-4 = “TU62”

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-4. This profile was developed by Tohru Uchida, who made this mouse. The double-banded reaction has not reliably worked, possibly because the band sizes are too similar. It is best to run these as two separate reactions.

Product Sizes

IRS-4: 300 WT, 350 Rec (approximately)