Preparation of Genomic DNA from Bacteria- using Phase Lock Gel TM

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Tony Rook
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Preparation of Genomic DNA from Bacteria- using Phase Lock Gel TM

Preparation of Genomic DNA from Bacteria- using Phase Lock Gel TM

(Modified from Experimental Techniques in Bacterial Genetics, Jones and Bartlet, 1990)

Posted at: http://www.research.umbc.edu/%7Ejwolf/m1.htm#genomic%20DNA

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 (Eppendorf-Brinkmann) (http://www.eppendorf.com/phaselockgel/en/phase_1.html)

METHOD:

1. Grow E. coli culture overnight in rich broth.
2. Transfer 2 ml to a 2-ml micro centrifuge tube and spin 2 min.
3. Decant the supernatant.
4. Drain well onto a Kimwipe.
5. Resuspend the pellet in 467 μl TE buffer by repeated pipetting.
6. Add 30 μl of 10% SDS and 3 μl of 20 mg/ml proteinase K, mix , and incubate 1 hr at 37 ° C.
7. Add an equal volume of phenol/chloroform and mix well but very gently to avoid shearing the DNA 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.
8. Carefully transfer the DNA/phenol mixture into a Phase Lock GelTM tube (green) and spin at 12,000 RPM for 10 min.
9. Transfer the upper aqueous phase to a new tube and add an equal volume of phenol/chloroform.
10. Again mix well and transfer to a new Phase Lock GelTM tube and spin 10 min.
11. Transfer the upper aqueous phase to a new tube.
12. Add 1/10 volume of sodium acetate. Mix.
13. Add 0.6 volumes of isopropanol and mix gently until the DNA precipitates.
14. Spool DNA onto a glass rod (or Pasteur pipet with a heat-sealed end).
15. Wash DNA by dipping end of rod into 1 ml of 70% ethanol for 30 sec.
16. Resuspend DNA in at least 200 μl TE buffer. Complete resuspension may take several days. Store DNA at 4 ° C short term, -20 or -80 ° C long term.
17. After DNA has dissolved, determing the concentration by measuring the absorbance at 260 nm.