ESC DNA prep

ES DNA Prep, Standard

Large Scale Isolation of DNA from ES Cells

1) Grow the ES cells on a 60 mm gelatin-coated dish , WITHOUT feeders, to near-confluence. This should be the second pass off feeders, otherwise, you may get considerable contamination with feeder DNA. 

2) Wash 2 times with PBS. The plates with the attached ES cells can now be stored at -20 or used directly for DNA isolation.

3) Add 2 ml of Laird lysis solution:

For 100 mls

              10 mM Tris 7.6           1 ml 1M
              10 mM EDTA               2 ml 0.5M
             0.5% Sarkosyl           2.5 ml 20%
              10 mM NaCl             0.2 ml 5M NaCl
             100 µg/ml Prot. K          Add fresh

............ wrap in parafilm and incubate 4 hours - O/N at 55 degrees.

4) Transfer contents (using transfer pipet, if you have them) to a 15 ml polypropylene screw-top tube. (Optional: Add 2 ml of TE if too viscous)

5) Do 1 phenol/chloroform extraction.

6) Add equal volume of isopropanol, invert several times to precipitate, then spool DNA out with Pasteur pipet which has a flame-closed end. Dip in 70% EtOH then 100% EtOH, and let pipet stand inverted for a few minutes.

7) Using a diamond-tipped pen, score off the portion of the Pasteur with the DNA into an Eppendorf tube containing 200-400µl of TE + 50 µg/ml RNase.

8) Rock O/N at 37 degrees. Use 15-30 µl in a digest for Southern blots.

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