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 by inversion using parafilm to cover the cuvette opening, replace the cuvette in the machine, and record the absorbance.

  
  

• 6. Repeat Step 5 using various volumes of Lambda Phage DNA standard added to the dye/buffer solution up to a cumulative added volume of 20 ml. Be sure to record the absorbance for the total volume (in ml) of Lambda Phage DNA added.

  
  

• 7. Thoroughly wash the cuvette with the dye/buffer solution prepared in Step 3. Shake as much excess dye/buffer solution from the cuvette as possible.

  
  

• 8. Place 2.00 ml of the dye/buffer solution (prepared in Step 3) in the cuvette, then add exactly 1.0 ml of the DNA to be quantitated, mix by inversion using parafilm to cover the cuvette opening, replace the cuvette in the machine, and record the absorbance. If the absorbance falls above the upper reading of the last standard measured, prepare an appropriate dilution of the DNA sample and repeat Steps 7 and 8. If the absorbance falls below the lower reading of the first standard measured, add one to several more ml of the sample until a reading is obtained. Be sure to record the volume of DNA sample used to obtain a particular absorbance reading.

  
  

• 9. Repeat Steps 7 and 8 for each DNA sample to be quantitated.

  
  

• 10. Using the data from Steps 5 and 6, prepare a standard curve to determine the concentrations (in mg/ml) from the absorbance readings from the DNA samples measured in Steps 7 through 9.

  
  

Appendix: Reagents/Solutions

Tween/albumin medium

• 1. Dissolve the following components in 995 ml of distilled-deionized water:

• Component Amount Added:

• KH2PO4 1.5 g

• Na2HPO4 . 2H20 1.9 g

• Na3citrate . 2H2O 0.4 g

• MgSO4 . 7H2O 0.05g

• CaCl2 . 2H2O (0.5 g/l) 1 ml

• ZnSO4 . 7H2O (0.5 g/l) 2 ml

• CuSO4 . 5H2O (0.5 g/l) 2 ml

• Sodium glutamate 0.5 g

• (NH4)2SO4 0.5 g

• Glycerol 2 ml

• NH4Fe(III)citrate (10 g/l) 1 ml

• Pyridoxine HCl (1 mg/ml) 1 ml

• Biotin (0.1 mg/ml) 5 ml

• Tween 80 (50 ml/l) 10 ml

• Albumin-glucose solution* 100 ml

• *This solution is made by dissolving 5 g of bovine albumin and 5 g of glucose in 100 ml of distilled-deoinized wate and adding 0.1 ml of a 100 g/l sucrose solution.

2. Filter sterilize the medium using a 0.22 mm membrane filter.

Solution A

• To 5.50 ml of TE Buffer (see below), add 10 mg of lysozyme. Mix thoroughly using a vortex. (NOTE: Not all the lysozyme will dissolve in the buffer.) Dispense 600 ml of the solution into 1.5 ml Eppendorf tubes and store frozen at -20 deg. C. Use one tube per strain being sure to mix the solution thoroughly prior to using. Discard the remaining solution.

Solution B

• 1. Prepare a 10 sodium lauryl sulfate (SDS; also known as sodium dodecyl sulfate) solution by dissolving 50 g SDS in 400 ml of distilled-deio nized wate. The solution will be cloudy, but adjust the pH to 7.2 using HCl. Bring the final volume to 500 ml with distilled-deionized water. This solution can be stored at room temperature without sterilization. (If the solution remains cloudy or becomes cloudy in the future, warm it to 50-65=B0C to dissolve the SDS before dispensing.)

  
  

• 2. Prepare a 10 mg/ml solution of proteinase K in distilled-deionized water. Dispense aliquots to 1.5 ml Eppendorf tubes and store at -20 deg. C. This solution can undergo several freeze-thaw cycles before the enzyme begins to deteriorate.

  
  

• 3. Solution B is made by mixing 700 ml of the 10 SDS solution with 60 ml of the proteinase K solution in a 1.5 ml Eppendorf tube. This solution can be stored at -20 deg. C and can undergo several freeze-thaw cycles before the enzyme begins to deteriorate.

  
  

5 M NaCl

• Dissolve 29.22 g of NaCl in 100 ml of distilled-deionized water. Store this solution at room temperature.

CTAB/NaCl

• Dissolve 4.1 g of NaCl in 80 ml of distilled-deionized water using a magentic stirrer and stir bar. While stirring, add 10 g CTAB (Hexzadecyltrimethylammonium bromide). If needed, dissolve the CTAB by heating the solution to 65 deg. C. Allow the solution to cool to room temperature. Adjust the final volume to 100 ml with distilled-deionized water.

Chloroform/Isoamyl alcohol

• Mix 96 ml chloroform with 4 ml isoamyly alcohol. Store this reagent at 4 deg. C.

TE Buffer, pH 8.0

• 1. Make a 1.0 M Tris buffer solution by adding 121.1 g of Trizma base (Tris) to 1.0 l of distilled-deionized water. Adjust the pH to 8.0 using HCl or NaOH. This solution can be sterilized by autoclaving and stored at room tempeature.

  
  

• 2. Make a 0.5 M EDTA solution by adding 186.1 g of Na2EDTA to 800 ml of distilled-deionized water. Adjust the pH to 8.0 by adding approximately 20 g NaOH pellets and mixing thoroughly. Make the final pH adjustments using a NaOH solution. Bring the final volume to 1.0 l with distilled-deionized water. This solution can be sterilized by autoclaving and stored at room temperature.

  
  

• 3. To 800 ml distilled-deionized water, add 10.0 ml of 1.0 M Tris, pH 8.0 and 2.0 ml 0.5 M EDTA, pH 8.0. Bring the total volume to 1.0 l with distilled-deionized water. If necessary, adjust the pH to 8.0 using HCl or NaOH. This solution can be sterilized by autoclaving and stored at room temperature. The final concentrations of the components are 10 mM Tris and 1 mM EDTA.

  
  

RNase

• Prepare DNase-free RNase by dissolving RNase A in a screw-cap tube at a concentration of 2 mg/ml in a buffer containing 10 mM Tris (pH 7.6) and 15 mM NaCl. Place the tube in a boiling water bath for 15 min, then allow it to slowly cool to room temperature. Dispense into small aliquots and store frozen at -20 deg C. Thaw just prior to use. A single aliquot of RNase can be used multiple times if refrozen and stored at -20 deg. C.

1X TNE Buffer

• 1. Prepare a 1.0 M Tris (pH 8.0) and a 0.5 M EDTA (pH 8.0) solutions as described for the TE buffer formulation given above.

  
  

• 2. To 900 ml distilled-deionized water, add 100 ml of 1.0 M Tris, pH 8.0 and 20 ml 0.5 M EDTA, pH 8.0. Dissolve 58.4 g NaCl in this solution and then bring the total volume to 1.0 l with distilled-deionized water. If necessary, adjust the pH to 8.0 using HCl or NaOH. This solution is considered to be 10X TNE and it can be stored at room temperature.

  
  

• 3. Prepare a 1X TNE buffer solution by diluting 10 ml 10X TNE to a final volume of 100 ml using distilled-deionized water.