PCR Genotyping of Mouse Tails for RIP/IRS-2 Tg


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 RIP/IRS-2 Tg band, use 5’FLAG and 3’Irs2T primers.


            5’FLAG:          ccg cca cca tgg act aca aag

            3’irs2T:            gac ggc tgt tcg caa ttg ag




PCR Mix Ingredients for single-band hot-start reaction


                        9.5 ml               water

                        2.0 ml               RedTaq 10X buffer

                        2.5 ml               DMSO

                        2.5 ml               primer 1 (previously diluted to 10pm/ml)

                        2.5 ml               primer 2 (previously diluted to 10pm/ml)

            Taq Mix

                        1.0 ml               dNTPs (previously diluted to 4 mM)

                        0.5 ml               RedTaq 10X Buffer

                        1.5 ml               H2O

                        2.0 ml               RedTaq polymerase


            Make up appropriate amount of Pre-Mix and add to PCR tubes containing 1 ml DNA.  Place in PCR machine and “incubate” at 95ºC for 5 minutes.  Remove tubes and add 5 ml of Taq mix to each tube.  Cap and return to PCR machine.  Run TU62 profile.


PCR Profile for IRS-3 = “RIPIRS2”


            95ºC                 5 minutes  [hot start]

            93ºC                 30 seconds

            62ºC                 30 seconds                                30 cycles

            72ºC                 2 minutes, 30 seconds

            72ºC                 5 minutes 

            4ºC                   forever



Product Sizes


            RIP/IRS-2 Tg:   1000 Tg (approximately)