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.
- Digest plasmid DNA (up to 20 µg) containing insert.
- Pour a gel containing the appropriate concentration of low-melting-
temperature agarous.
- Mix the samples of DNA with tracking dye, heat shock at 65
C for 5 min, transfer in ice, and load onto the gel.
- 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.
- Take a picture.
- 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.
- Add appr. 5 volumes of H2O to the slice of agarous.
- 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
- Spin the tubes (4K, 10 min, 20C).
- Save the supernatent with Micropipet (P-20) into new MFT.
The white substance at the interphase is powdered agarous.
- Re-extract the agarous phase once with phenol:chloroform and
once with chloroform.
- 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.
- Centrifuge 5 K, 20 min at 4 C.
- Dump supernatant, rinse with 70% EtOH, dry briefly, and resuspended
in 5 to 10 µl TE.
- 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.