of Genomic DNA in Agarose Plugs
Van Ommen, G.J.B. and J.M.H. Verkerk
(1986) in Human Genetic Diseases, A Practical Approach,
pp113-132, K.E. Davies (ed.), IRL Press Ltd., Oxford,
Begin with counted cells that
have been wash in PBS to remove serum, trypsin,
and/or RBC lysate.
Wash twice with 10ml PBS.
Resuspend to approximately 1 x
107 cells/ml in PBS (this gives about
0.4 to 0.5 million cells per plug).
Warm solution to 50oC.
Make "InCert" agarose at 1.2%
in SE buffer (be sure to weigh suspension before
and after boiling to replace water lost by evaporation).
Cool 1.2% agarose to 50oC
in a waterbath.
Mix with an equal volume of cells
to give 0.6% agarose suspension.
Dispense into taped-up molds (about
80Ál per slot).
Place at 4oC for 30
Using a rubber bulb, blow plugs
out of slots into 50ml tubes containing 5ml ESP
(one tube for ~20 plugs).
Incubate tubes at 50oC
for ~48 hours.
Decant ESP and wash plugs twice
(for at least one hour each time) at room temperature
with 50ml TE containing 1 mM PMSF (phenyl-methyl-sulfonyl-fluoride).
Wash three times with TE without
Store at 4oC in 10ml
0.5M EDTA pH 8.0
SE: 75 mM NaCl 25mM EDTA (pH 7.4)
TE: 10 mM Tris-HCl (pH 8.0) 1 mM EDTA
ESP: Dissolve 1 gram SDS in 100 ml 0.5M EDTA
(pH 9.0). Add 100mg proteinase K. Store at -20oC
in 5ml aliquots.