PCR Genotyping of Mouse Tails for IRR

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: (see Matt for proper primer names)

To assess IRR endogenous band, use 4857 and 4856 primers.

To assess IRR neo band, use 4856 and 3202 primers.

To assess IRR complete double-band reaction, use 4856, 4857, and 3202 primers.

IRR 4856: gtg tgt ccc tgc ccc cga ggg

IRR 4857: tga cac agc gcc agg act cat

IRR 3202: cgc ctt ctt gac gag ttc ttc tg

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 IRR 4856, to ensure that there is enough of these two primers to anneal with both the 4857 and 3202 primers.

PCR Profile for IRR

94ºC 3 minutes

94ºC 1 minute

56ºC 1 minute 35 cycles

72ºC 2 minutes

72ºC 5 minutes

4ºC forever

This profile may have changed (this was the original one we received from Serge). See Matt for any updates. See Matt for profile name.

Product Sizes

IRR: 250 WT, 550 Rec (approximately)