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Materials:
- TE buffer
- 10% (w/v) sodium
dodecyl sulfate (SDS)
- 20 mg/ml proteinase
K
- phenol\chloroform
- isopropanol
- 70% ethanol
- 3M sodium acetate
pH 5.2
- Phase Lock GelTM
(5 Prime, 3 Prime, Inc)
1. Transfer 1.5 ml to
a micro centrifuge tube and spin 2 min. Decant the supernatant.
Drain well onto a Kimwipe.
2. Resuspend the pellet in 467 µl TE buffer by repeated pipetting.
Add 30 µl of 10% SDS and 3 µl of 20
mg/ml proteinase K, mix , and incubate 1 hr at 37 C.
3. Add an equal volume of phenol/chloroform and mix well by inverting
the tube until the phases
are completely mixed. CAUTION:
PHENOL CAUSES SEVERE BURNS, WEAR GLOVES GOGGLES, AND LAB
COAT AND KEEP TUBES CAPPED TIGHTLY. Carefully transfer the DNA/phenol
mixture into a Phase Lock GelTM tube(green)
and spin 2 min.
4. Transfer the upper aqueous phase to a new tube and add an equal volume
of phenol/ chloroform. Again mix well and transfer
to a new Phase Lock GelTM tube and spin 2 min. Transfer
the upper aqueous phase to a new tube.
5. Add 1/10 volume of sodium acetate.
6. Add 0.6 volumes of isopropanol and mix gently until the DNA
precipitates.
7. Spool DNA onto a glass rod (or Pasteur pipet with a heat-sealed
end).
8. Wash DNA by dipping end of rod into 1 ml of 70% ethanol for
30 sec.
9. Resuspend DNA in 100-200 µl TE buffer.
10. After DNA has dissolved, measure the concentration by diluting
10 µl of DNA into 1 ml of TE (1:100 dilution)
and measure absorbance at 260 nm.
Concentration of original DNA solution in µg/ml
= Abs x 100 x 50 µg/ml.
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