PCR Genotyping of Mouse Tails for IRS-3

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-3 endogenous band, use IRS-3 ko 1 and IRS-3 ko 2 primers.

To assess IRS-3 neo band, use IRS-3 ko 1 and IRS neo primers.

To assess IRS-3 complete double-band reaction, use IRS-3 ko 1, ko 2, and IRS neo primers.

IRS-3 ko 1: gta gga acg gac agt gct agg ctg

IRS-3 ko 2: ttc cta ggt ggt aca gct gga agg

IRS neo: gct acc cgt gat att gct gaa gag

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-3 ko 1, to ensure that there is enough of this primer to anneal with both the ko 2 and neo primers.

PCR Profile for IRS-3 = “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-3. This profile was developed by Tohru Uchida, who made this mouse.

Product Sizes

IRS-3: 300WT, 350 Rec (approximately)