Preparation of PFGE-quality DNA from members of the Mycobacterium tuberculosis complex

 

Preparation of intact chromosomal DNA is of central importance to all genomics projects. All of the BAC libraries generated at GMB, and the maps and restriction profiles obtained from pulsed field gel electrophoresis (PFGE) have used DNA prepared by the following protocol:

Growth and Harvesting:

From a 7-day starter culture, inoculate 100-300 ml of 7H9/ADC.

Let culture grow for 7-10 days (the culture should be in the exponential phase).

Add glycine to a final concentration of 0.2M and leave for 18-24 hours.

Harvest cells at 4000g for 20 minutes.

Inactivate virulent strains by heating the pellet for 20 minutes at 75 degrees Celsius

Wash the pellet in 200 ml of ice-cold TE buffer; recentrifuge at 4000g for 30 minutes (pellet will be delicate so decant the supernatant off very carefully).

 

Dilutions:

Resuspend the pellet (approx. 1 g wet weight cells) in 5 ml of TE (ice-cold).

Dilute the suspension 1:2. This is the High concentration.

Stepwise dilute the suspension 1:5 (i.e. 1:10 of original, this is the Medium concentration), and dilute this again 1:2 (i.e. 1:20 of original; this is the Low concentration).

 

Plugs:

For each concentration of cells, take 900 microliters of the suspension and add 900 microliters of molten 1% Low melting point agarose (made in 1 x TE, pH 8; autoclave to dissolve).

Pipette well to mix, then add 85 microliters of the suspension to Biorad disposable plug molds (cat no. 170-3713).

Let the agarose set on ice for 10-20 minutes.

 

DNA Release:

Push plugs into TNE buffer: 2 x TNE buffer 25 ml

10% sarkosyl 2.5 ml

Sterile distilled water to 50 ml

Lysozyme 60 mg

(Don’t use more than 50 plugs per 50 ml)

Incubate overnight at 37°C.

Decant the solution (it’s easier to do this against black background to see the plugs), wash the plugs at 4°C in 50 ml of 1x TNE, 0.5 % sarkosyl for 30 minutes.

Incubate at 50°C for 24 hours in ESP solution.

Decant the solution and add fresh ESP solution; continue the incubation for a further 24 hours at 50°C.

Wash the plugs in 30 ml of 1 x TE at 4°C for 30 minutes.

Decant the solution, add 30 ml of TE and preheat the solution at 50°C for 15 minutes. Add 30 (or up to 300) microliters of PMSF solution; incubate at 50°C for 1 hour.

Wash the plugs in 50 ml 0.2 M EDTA (pH 8.5) at 4°C for 30 minutes.

Store in 30 ml 0.2M EDTA (pH 8.5).

 

Restriction endonuclease digestions:

Wash the plugs in sterile 0.01% Triton X-100 (2 ml per plug) at 4°C for 1 hour to overnight.

Wash the plugs at room temperature in 1 x enzyme buffer (2 ml per plug) for 30 minutes.

Digest the plugs in 1 x buffer with 20-30 Units of enzyme for 4 hours to overnight.

Stop the reaction by adding 1 ml of 50 mM EDTA (pH 8.0).

 

Solutions:

2 x TNE Buffer: 12 mM Tris-HCl, 2 M NaCl, 0.2 M EDTA


To prepare this 2X buffer use 1,2 ml of 1M Tris-HCl pH 7.6, 40 ml 5M NaCl, 40 ml 0.5M EDTA pH 8.5 and 18,8 ml H20 to get 100 ml of stock solution.


ESP: 30 ml 0.5 M EDTA (pH 8.5)

1.5 ml 10% sarkosyl

60 mg Proteinase K.



PMSF: 40 mg phenyl methyl sulphonate in 1 ml isopropanol


 

Trouble-shooting:

We routinely use plugs at three different DNA concentrations for reasons of convenience. If the samples are too concentrated they will yield poor quality DNA, probably because the ESP treatment is inadequate. In this case, plugs often have a gritty aspect. Plugs generally become clear after successful ESP treatment. On occasion, the DNA can be smeary and quality tends to vary from strain to strain for reasons that we don’t understand. In our hands better results are achieved with M. tuberculosis, then BCG, and finally M. bovis. To eliminate smeariness, try repeating the ESP treatment for a further 24 h. If this doesn’t improve things, repeat the culture but harvest at an earlier stage in the growth cycle. Cycloserine can be used to weaken cell walls instead of glycine but is more expensive and can provoke lysis. The use of both glycine and cycloserine is not recommended.


Please address questions and comments to Stephen GORDON, (present address: United Kingdom's Veterinary Laboratories Agency) or to Roland BROSCH

DISCLAIMER The information contained in this webpage is provided in good faith, but no warranty, nor any legal liability or responsibility for any purpose for which the data are used, is given.

Institut Pasteur UnitM-i de GM-inM-itique MolM-iculaire