PCR Genotyping of Mouse Tails for PDX KO and PDX Tg

 

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 95C).

 

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 55C water bath, room 635 on Liangyous 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 95C 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 dont 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 PDX endogenous band, use PDXHD1R and PDXHD1F primers.

To assess PDX neo band, use SD 54 lacz and SD 53 primers.

To assess PDX Tg band, use PDXTg (R) and PDXTg (F) primers.

 

PDXHD1R: agc tcc tcg ccc gag gtc ac

PDXHD1F: gct gct gga gct gga gaa gg

SD 54 lacz: acc acc acg ctc atc gat aat

SD 53: aac agt tgc gca gcc tga atg

 

PDXTg (R): ggg ggt gca act gtt gaa gaa

PDXTg (F): cag tcg gac gta tag gga aac

 

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 Profile for PDX (neo) and PDX Tg = PDX

 

94C 3 minutes

93C 30 seconds

52C 30 seconds 35 cycles

72C 1 minute

72C 10 minutes

4C forever

 

PCR Profile for PDX (endo) = PDXENDO

 

94C 3 minutes

93C 30 seconds

70C 30 seconds 35 cycles

72C 1 minute

72C 10 minutes

4C forever

 

The double-banded reaction for PDX (endo and neo) obviously does not work because of the different annealing temperatures.

 

Product Sizes

 

PDX: 270 WT, 600 Rec

PDX Tg: 210 Tg