PCR Genotyping of Mouse Tails for IGFR


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 IGFR neo band, use IGFR 5’ and IGFR 3’ primers.


            IGFR 5’:          cag gac ata gcg ttg gct acc c

            IGFR 3’:          gga cct tct aca agg tgg gga c



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 IGFR = “IGF”


            94ºC                 1 minute

            94ºC                 30 seconds

            60ºC                 30 seconds                                30 cycles

            72ºC                 1 minute, 30 seconds

            72ºC                 10 minutes 

            4ºC                   forever



Product Sizes


            IGFR:   1000 Rec


We have been unsuccessful in designing primers to assess the endogenous band for these mice.  Conveniently, IGFR knockout mice die at birth, which means that any weaned mice are either heterozygous or wild-type.  However, on occasion, someone will take embryos or dead pups for experimentation and will want to know their genotype.  For this we must use the Southern blot technique.