Plasmid DNA Isolation from Bacteria

 Materials:

TE buffer

10% (w/v) sodium dodecyl sulfate (SDS)

20 mg/ml proteinase K

phenol\chloroform (50:50)

isopropanol

70% ethanol

3M sodium acetate pH 5.2

Phase Lock GelTM (5 Prime, 3 Prime, Inc)

1.Grow E. coli culture overnight in rich broth. Transfer 1.5ml to a micro centrifuge tube and spin 2min. Decant the supernatant. Repeat with another 1.5ml of cells. Drain well onto a Kimwipe.

2.Resuspend the pellet in 467μl TE buffer by repeated pipetting. Add 30μl of 10% SDS and 3μl of 20mg/ml proteinase K, mix , and incubate 1 hr at 37EC.

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 2min.

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 5 min. Transfer the upper aqueous phase to a new tube.

5.Add 1/10 volume of sodium acetate. Mix.

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 1ml of 70% ethanol for 30 sec.

9. Resuspend DNA in 100-200μl TE buffer. Complete resuspension may take several days.

10. Store DNA at 4EC short term, -20 or -80EC long term

11. After DNA has dissolved, determing the concentration by measuring the absorbance at 260nm.