PREPARATION OF GENOMIC DNA FROM BACTERIA

Solutions required for this protocol

· TE buffer
· 10% (w/v) sodium dodecyl sulfate (SDS)
· 20 mg/ml proteinase K
· Phenol/chloroform
· Isopropanol
· 70% ethanol
· 3M sodium acetate ph 5.2
· Phase Lock geltm (5 Prime, 3 Prime, Inc)

1. Transfer 1.5 ml to a micro centrifuge tube and spin 2 min. Decant the supernatant. 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°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).
Wash DNA by dipping end of rod into 1 ml of 70% ethanol for 30 sec.
Resuspend DNA in 100-200 µl TE buffer.

8. 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.

Protocol of Genomic DNA Isolation from a Gram-Negative Bacterium

The G NOME® kit (http://www.qbiogene.com) is used to quickly and efficiently isolate high molecular weight genomic DNA from Gram-negative bacterial cultures. Each preparation with the G NOME® kit yields up to 100 mg of genomic DNA. The DNA isolated by the G NOME® procedure is suitable for restriction enzyme digestion or PCR amplification in as little as 1 hour after cell lysis.

Protocol

1. Bring cells*/tissues to a final volume of 1.85ml in Cell Suspension Solution. (Use a 15 ml clear plastic tube for efficient mixing). Mix until the solution appears homogeneous.

2. Add 50ml of RNase Mixx, mix thoroughly.

3. Add 100ml of Cell Lysis/Denaturing Solution**, mix well.

4. Incubate at 55°C for 15 minutes.

5. Add 25ml Protease Mixx, mix thoroughly. (Note: If precipitate is visible in Protease Mixx suspension, pulse spin and use 25 ml of supernatant.)

6. Incubate at 55°C for 30 to 120 minutes. (The longer time will result in minimal protein carry over and will also allow for substantial reduction in residual protease activity.)

7. Add 500ml “Salt-Out” Mixture, mix gently yet thoroughly. Divide sample into 1.5ml tubes. Refrigerate at 4°C for 10 minutes.

8. Spin for 10 minutes at maximum speed in a microcentrifuge (at least 10,000 x g). Carefully collect the supernatant, avoid the pellet. If a precipitate remains in the supernatant, spin again until it is clear. Pool the supernatants in a 15 ml (or larger) clear plastic tube.

9. To this supernatant, add 2 ml TE buffer and mix. Then add 8mls of 100% ethanol. If spooling the DNA, add the ethanol slowly and spool the DNA at the interphase with a clean glass rod. If centrifuging the DNA, add the ethanol and gently mix the solution by inverting the tube. Spin for 15 minutes at 1000-1500xg. Eliminate the excess ethanol by blotting or air drying the DNA.

10. Dissolve the genomic DNA in TE (10mM Tris pH 7.5, 1mM EDTA).