PCR Genotyping of Mouse Tails for JNK1, JNK2, and JNK3

 

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 JNK1 endogenous band, use JNK1-MJ1F8 and JNK1-MJ1B8 primers.

To assess JNK1 neo band, use JNK1-MJ1F8 and JNK1-PGKT1 primers.

To assess JNK1 complete double-band reaction, use JNK1-MJ1F8, JNK1-MJIB8, and JNK1-PGKT1 primers.

To assess JNK2 endogenous band, use JNK2-MJ2B3 and JNK2-MJ3F5 primers.

To assess JNK2 neo band, use JNK2-MJ2F5 and JNK1-PGKT1 primers.

To assess JNK2 complete double-band reaction, use JNK2-MJ2B3, JNK2-MJ3F5, and JNK1-PGKT1.

To assess JNK3 endogenous band, use JNK3-MJ3B3 and JNK3-MJ3F3 primers.

To assess JNK3 neo band, use JNK3-MJ3F3 and JNK3-PGKP1 primers.

To assess JNK3 complete double-band reaction, use JNK3-MJ3B3, JNK3-MJ3F3, and JNK3-PGKP1 primers.

 

            JNK1 MJ1B8:             cgc cag tcc aaa atc aag aat c

            JNK1 MJ1F8:              gcc att ctg gta gag gaa gtt tct c

            JNK1 PGKT1:             cca gct cat tcc tcc act cat g

 

            JNK2 MJ2B3:             gga gcc cga tag tat cga gtt acc

            JNK2 MJ2F5:              gtt aga caa tcc cag agg ttg tgt g

            JNK1 PGKT1:             cca gct cat tcc tcc act cat g

 

            JNK3 MJ3B3:             cct gct tct cag aaa cac cct tc

            JNK3 MJ3F3:              cgt aat ctt gtc aca gaa atc cca tac

            JNK3 PGKP1:             ctc cag act gcc ttg cca aaa

 

The following primer mixes are easier to use than the individual primers.  These can be made and stored in the cold room.

 

JNK1 Primer Mix

            200 ml MJ1B8 (100 mM)

            200 ml MJ1F8 (100 mM)

            200 ml PGKT1 (100 mM)

            730 ml ddH2O

JNK2 Primer Mix

            100 ml MJ2B3 (100 mM)

            200 ml MJ2F5 (100 mM)

            200 ml PGKT1 (100 mM)

            300 ml ddH2O

JNK3 Primer Mix

            200 ml MJ3B3 (100 mM)

            200 ml MJ3F3 (100 mM)

            200 ml PGKP1 (100 mM)

            730 ml ddH2O

 

PCR Mix Ingredients for double-band reaction

            18.5 ml             water

            2.5 ml               RedTaq 10X buffer

            1.5 ml               correct primer mix

            1.25 ml             dNTPs (previously diluted to 4 mM)

            0.25 ml             RedTaq polymerase (5 U/ml)

 

 

PCR Profile for JNK1, JNK2 and JNK3 = “JNK”

 

            94ºC                 2 minutes

            94ºC                 30 seconds

            56ºC                 30 seconds                                29 cycles

            72ºC                 1 minute

            end

 

I have tried these protocols several times with varying degrees of success.  I can see both bands for JNK2 quite clearly and am confident these are correct.  However, I usually only get one band for JNK1 and JNK3.  I have knockout tail DNA from each JNK and you should always use wild-type DNA (tail DNA from any other cross) to confirm whether you’re seeing the correct bands.

 

 

Product Sizes

 

            JNK1:   460 WT, 390 Rec

            JNK2:   400 WT, 270 Rec

            JNK3:  430 WT, 250 Rec