Preparation of Genomic DNA from Bacteria- using Phase Lock GelTM
(Modified from Experimental Techniques in Bacterial Genetics, Jones and Bartlet, 1990)
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TE buffer |
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10% (w/v) sodium dodecyl sulfate (SDS) |
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20 mg/ml proteinase K |
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phenol\chloroform (50:50) |
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isopropanol |
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70% ethanol |
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3M sodium acetate pH 5.2 |
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Phase Lock GelTM (5 Prime, 3 Prime, Inc)
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 37 EC.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 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 4 EC short term, -20 or -80EC long term11. After DNA has dissolved, determing the concentration by measuring the absorbance at 260 nm. |
