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Recovery of DNA from low-melting-temperature agarous gels

Caution: Ultraviolet radiation and EtBr in a gel are dangerous. Wear protective goggles and gloves to protect the eyes and EtBr contamination.

  1. Digest plasmid DNA (up to 20 µg) containing insert.
  2. Pour a gel containing the appropriate concentration of low-melting- temperature agarous.
  3. Mix the samples of DNA with tracking dye, heat shock at 65 C for 5 min, transfer in ice, and load onto the gel.
  4. Carry out electrophoresis at 12 volt overnight.
    * DNA of a given size runs slightly faster through gels cast with low- melting-temperature agarous than through conventional gels.
  5. Take a picture.
  6. Using a hand-held UV light with long wavelength (to minimize the damage to the DNA), cut out insert bands using a scalpel. Cut the gel as close to band of interest as possible and transfer it to a clean 1.5 ml MFT. Check or draw the removing band on the picture for a record of which band was eluted.
  7. Add appr. 5 volumes of H2O to the slice of agarous.
  8. Melt the agarous at 65 C for 1 to 5 min. Voltex for 20 sec and store at -20 C.

    For further separation of DNA from agarous

  9. Spin the tubes (4K, 10 min, 20C).
  10. Save the supernatent with Micropipet (P-20) into new MFT. The white substance at the interphase is powdered agarous.
  11. Re-extract the agarous phase once with phenol:chloroform and once with chloroform.
  12. Transfer the aqueous phase to a MFT, bring up to 105 µl with H2O, add 35 µl NH4Ac and 1050 µl EtOH, and invert to mix.
  13. Centrifuge 5 K, 20 min at 4 C.
  14. Dump supernatant, rinse with 70% EtOH, dry briefly, and resuspended in 5 to 10 µl TE.
  15. Store at -20 C

If further purification of DNA is necessary, pass the DNA through DEAE-Sephacel.

Reference: Sambrooks et al. 1989. P6.30, 6.31. [an error occurred while processing this directive]