PCR Genotyping of Mouse Tails for PTP-1B


Make up stock solution of digestion buffer, leaving out b-ME.


Digestion Buffer



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 PTP-1B endogenous band, use PTP1B endogenous left and PTP1B endogenous right #3 (ERP3) primers.

To assess PTP-1B neo band, use PTP1B endogenous left and PTP1B neo primers.

To assess PTP-1B complete double-band reaction, use PTP1B endo right 3, endo left, and neo primers.


            PTP1B endo left:          cag tct tgg tct aca gag tg

            PTP1B ERP3:              cga tct cct cga act cct tc

            PTP1B neo:                  gcg agc tgt gga aaa aaa agg


PCR Mix Ingredients for double-band reaction

            Pre-Mix (14.75 ml)

                        9.83 ml             water

                        2.0 ml               RedTaq 10X buffer

                        2.5 ml               DMSO

                        0.24 ml             primer 1 (endogenous left) (previously diluted to 20pm/ml)

                        0.12 ml             primer 2 (endogenous right #3) (previously diluted to 20 pm/ml)

                        0.06 ml             primer 3 (neo) (previously diluted to 20pm/ml)

            Taq Mix (9.25 ml)

                        0.5 ml               RedTaq 10X Buffer

                        1.875 ml           water

                        6.25 ml             dNTPs (previously diluted to 4 mM)

                        0.625 ml           RedTaq polymerase


Make up pre-mix and add 14.75 ml to each PCR tube containing 1 ml DNA.  Run the “incubate” program on the PCR machine at 94ºC for 3 minutes.  When this has finished, remove one row of tubes (they will be pretty hot) and start the PTP1B program.  Add 9.25 ml of the Taq mix to each tube, cape the tubes and place them back in the PCR machine.  This will keep them heated at 82ºC until the Taq mix has been added to all the tubes.  This should not take more than the 10 minutes allotted to this step of the profile.  Place all the capped tubes back in the machine and put the cover on.  Fast-forward from the 82ºC step to the next step and let the program run.


PCR Profile for PTP-1B = “PTP1B”


            94ºC                 3 minute

            82ºC                 10 minutes (or as long as it takes to add Taq mixture)

            93ºC                 15 seconds

            60ºC                 30 seconds                                40 cycles

            65ºC                 2 minutes, 30 seconds

            65ºC                 10 minutes 

            4ºC                   forever


The original PTP1B endogenous right primer was: atc tcc tcg aac tcc ttc tc.  However, we could not get the endogenous band to appear.  So Jake made three new sets of primers to substitute and we tried each.  The primer sequence listed above (ERP3) was the one that worked best.  Since that time, this has been a difficult gene to PCR.  Sometimes it works, sometimes it doesn’t.  Thankfully, we haven’t had to deal much with it recently since the mice are in holding at Charles River.  However, expect occasional problems with genotyping once the rederived pups are tailed.  The original program was also for a 52ºC annealing temperature.



Product Sizes


            PTP1B:            1000 WT, 300 Rec