Isolation and Quantification of Genomic DNA from Mycobacterium tuberculosis

Part A. Isolation of Nucleic Acids
NOTE: CAUTION! STEPS 1-10 SHOULD BE PERFORMED USING APPROPRIATE PROCEDURES FOR HANDLING MATERIAL POTENTIALLY CONTAMINATED WITH MYCOBACTERIUM TUBERCULOSIS. ALSO,THE LIDS OF THE EPPENDORF TUBES SHOULD BE CAREFULLY CLOSED AND OPENED SO AS TO AVOID SPLASHES AND AEROSOL =46ORMATION.1. Using a 1ml plastic, disposable pipet attached to a Pipet-Aid motorized pipettor, add 1 ml sterile TE buffer to an L&J slant containing MTB colonies selected for extraction of
DNA
. Using the end of the pipet, dislodge colonies from surface of medium until all colonies are suspended in the 1 ml TE buffer. Be careful not to disrupt the surface of the medium. Remove the suspended cells in the TE buffer to a 1.5 ml, sterile, screw-capped microfuge tube and seal tube.2. Place the sealed microfuge tube in an 80 deg. C oven for 60 min.3. Centrifuge the Eppendorf tube for 5 min at room temperature using an aerosol-containing microfuge operating at 9,000 rpm.4. Carefully remove the supernatant using a disposable, cotton-plugged Pasteur pipette. Discard the pipette in an appropriate manner.5. To the remaining cell pellet, add 550 ml of Solution A. Thoroughly resuspend the pellet using a vortex. Incubate the cell suspension at 37 deg. C for 1 hour.6. To the cell suspension, now add 76 ml of Solution B. Thoroughly mix the contents of the Eppendorf tube using a vortex. Incubate the cell suspension at 65 deg. C for 10 min.7. Next, add 100 ml of 5 M NaCl to the cell suspension and thoroughly mix the contents of the Eppendorf tube using a vortex. Then add 80 ml of CTAB/NaCl and again thoroughly mix the contents of the Eppendorf tube using a vortex. Incubate the resulting suspension at 65 deg. C for 10 min.8. After the above incubation step, add 700 ml of chloroform/isoamyl alcohol. Thoroughly mix the contents of the Eppendorf tube at least 15 sec using a vortex. Then, centrifuge the Eppendorf tube for 5 min at room temperature using a microfuge operating at 14,000 rpm (@15,300 x g).9. Using a disposable pipette, remove the upper aqueous layer (without disturbing or carrying over any of the white middle layer) to a second 1.5 ml Eppendorf tube. Fill the remaining volume of the Eppendorf tube with isopropanol, seal the tube, and invert it several times to mix the contents. Incubate the tube at -20 deg. C at least 30 min.
NOTE: After adding the isopropanol, the Eppendorf tubes can be stored at -20 deg. C until transported to the general microbiology laboratory or they can be immediately taken to the General microbiology laboratory for theincubation at -20 deg. C and subsequent handling.10. Collect the nucleic acids by centrifugation for 30 min using a microfuge operating at 14,000 rpm (@15,300 x g). Gently drain off the supernatant, then carefully add approximately 1 ml of cold 70 (v/v) ethanol. Again, collect the nucleic acids by centrifugation for 15 min using a microfuge operating at 14,000 rpm (@15,300 x g).11. Carefully drain off the supernatant and evaporate the remaining ethanol using the Speed-Vac Concentrator for 30 min.12. Dissolve the nucleic acid pellet in 50 ml TE Buffer. Be sure to dissolve any of the precipitate adhering to the "spine" of the Eppendorf tube by washing it with the TE buffer.13. Optional Step: To remove contaminating RNA from the preparation, add 1 ml of RNase to the nucleic acid solution. Incubate the tube at 37 deg. C for 30 min.
NOTE: The nucleic acid solution should be stored at -20 deg. C when not in = use.Part B. Determination of DNA Concentration 1. Turn on the Hoeffer DNA Fluorimeter and allow it to warm up at least 30 min. (NOTE: If the sample was stored frozen, allow it to thaw at room temperature. Once thawed, keep the sample on ice until it is processed.) 2. Prepare a 25 ng/ml solution of Lambda Phage DNA to be used as a standard for determining the concentration of DNA derived from the various M. tuberculosis strains. This is done by diluting 20 ml of Lambda Phage DNA (at a concentration of 0.25 mg/ml) with 180 ml of sterile, distilled-deionized water. This standard solution can now be stored frozen at -20 deg. C until needed. Also, it can be repeatedly frozen/thawed and kept on ice during use. 3. Pass 100 ml of 1X TNE buffer through a 0.22 mm filter to remove any particulate matter. To this buffer, add 10 ml of Hoescht dye 33257 (1 mg/ml)=DD to obtain a final dye concentration of 100 ng/ml. 4. Place 2.00 ml of the dye/buffer solution (prepared in Step 3) in the cuvette. Zero the fluorimeter. 5. Remove the cuvette, add exactly 1.0 ml of the Lambda Phage DNA standard, mix