MOLECULAR BIOLOGY PROTOCOLS
M.1: Preparation of genomic DNA from bacteria M.2: PCR amplification of DNA M.3: Restriction enzyme digestion of DNA M.4: Phenol/chloroform extraction of DNA M.5: Ethanol precipitation of DNA M.6: Agarose gel electrophoresis M.7: Transformation of E. coli by electroporation M.8: Wizard PCR preps DNA purification system M.9: Alternate method for purifying DNA from agarose gels M.10: Transfection of mammalian cells using Lipofectamine (LTI) M.11: Southern blotting M.12: RT-PCR Protocol M.13: Preparation of sequencing gels M.14: Isolation of RNA using RNAzolTM
M.1: PREPARATION OF GENOMIC DNA FROM BACTERIA
(Modified from Experimental Techniques in Bacterial Genetics, Jones and Bartlet, 1990)
1. Transfer 1.5 ml to a micro centrifuge tube and spin 2 min. Decant the supernatant. Drain well onto a Kimwipe.
- TE buffer
- 10% (w/v) sodium dodecyl sulfate (SDS)
- 20 mg/ml proteinase K
- 70% ethanol
- 3M sodium acetate pH 5.2
- Phase Lock GelTM (5 Prime, 3 Prime, Inc)
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.