Preparation of Genomic DNA from Bacteria- using Phase Lock GelTM

1. Grow E. coli culture overnight in rich broth. Transfer 1.5 ml to a micro centrifuge tube and spin 2 min. Decant the supernatant. Repeat with another 1.5 ml of cells. 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 37E 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 Gel^TM 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 Gel^TM 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 1 ml of 70% ethanol for 30 sec.

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

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

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