Preparation 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, England

1. Begin with counted cells that have been wash in PBS to remove serum, trypsin, and/or RBC lysate.
2. Wash twice with 10ml PBS.
3. Resuspend to approximately 1 x 10⁷ cells/ml in PBS (this gives about 0.4 to 0.5 million cells per plug).
4. Warm solution to 50^oC.
5. Make "InCert" agarose at 1.2% in SE buffer (be sure to weigh suspension before and after boiling to replace water lost by evaporation).
6. Cool 1.2% agarose to 50^oC in a waterbath.
7. Mix with an equal volume of cells to give 0.6% agarose suspension.
8. Dispense into taped-up molds (about 80µl per slot).
9. Place at 4^oC for 30 minutes.
10. Using a rubber bulb, blow plugs out of slots into 50ml tubes containing 5ml ESP (one tube for ~20 plugs).
11. Incubate tubes at 50^oC for ~48 hours.
12. 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).
13. Wash three times with TE without PMSF.
14. Store at 4^oC 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 -20^oC in 5ml aliquots.