I.  Do tail snips.  Lightly anaesthetize mice with isofluorane, so they are woozy but not out cold.  Hold them firmly behind the ears.  Dip tail in ice water.  Lop off 1/4 inch with clean razor.  Also do ear punch to later identify the mice after you’ve done the PCR and know which are keepers.  While you have them, try to sex them.  Put mice in separate cage to recover and stop bleeding.

Put tail snip into 100 ul of "PCR homog buffer" (10X stock in 15 ml conical in refrig rm 211.  1X stock also in refrig, labeled "tail buffer").  Add 10 ul 20 mg/ml Prot K.  Leave in 55 deg water bath O/N.  Tail will more or less dissolve, but bits of bone and hair may remain.

II.  Do the PCR.  You do not need to ph/cl this DNA.  Boil DNA 10 min., then spin out crud (see above).  Use 5 to 10 ul of each tail prep for PCR.  Start with 5 and if you don’t get any bands, try 10.  Since we are looking to identify hets, use the neo primers, as follows:  Stuff is all in my box "mice" in Sebbie.  Use some old het DNA as positive control.  The original male (circled arrow) is good.

5-10 ul tail DNA
4 ul dNTPs
1 ul each primer, Ihh5’ and PGKneo
5 ul Sigma 10x
H2O to 50 ul
Add 0.5 ul taq hot start, after 5 min denature.

Program "neotail"

Run gel.  You are looking for a 300 bp band.  I usually get some stray bands.  Run a 1.0% gel so you can easily match up to  old ones when you are trying to find the real band.

10X PCR homog  buffer (from p. 303 in mouse book)

500mM  KCl
100 mM Tris pH 8.3
0.1 mg/ml gelatin (tissue culture stuff is OK)
0.45% NP-40
0.45% Tween 20

After you have the tails in the 1X solution, add 500 ug/ml Prot K, from stock.