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HOME > Protocols > Molecular Biology > DNA > Protocol for Preparation of Genomic DNA in Agarose Plugs

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 50oC.
  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 50oC in a waterbath.
  7. Mix with an equal volume of cells to give 0.6% agarose suspension.
  8. Dispense into taped-up molds (about 80Ál per slot).
  9. Place at 4oC 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 50oC 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 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.

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