DNA isolation SOP

Glenn Wall-Manning gmanning at wnmeds.ac.nz
Wed Aug 19 16:44:00 EST 1998


I have been using this method for a while, and it seems to give good results
with a wide variety of gram pos and neg bacteria.  It's a mix of a number of
methods, so I can't really reference it.  The key point is to catch the
exponential cultures early in their growth phase.  I normally let them grow
to about OD600 0.4.  I use TSBYK media because all my bugs grow in it, and I
only have to make up one media type ;-), but use whatever you use to grow
your bugs.  Good luck, and if you want to discuss it further, email me at
gwallmanning at wnmeds.ac.nz

Inoculate 4 mL TSBYK from fresh plate of cells.  Grow overnight at 35C under
appropriate gas conditions.
Inoculate 40 mL TSBYK with overnight culture.  Grow, with shaking, at 35C
until OD600 reaches no more than 0.8.  (Gram positive bacteria:  Adjust the
pH of aerobes to pH 7.0.   Add 50 uL (8000 units) penicillin G, and grow for
a further hour).
Harvest, and resuspend in 3 mL of SET buffer.  Add 50 uL of RNaseA (10
mg/mL) and 2 mg lysozyme.  Incubate at 37C overnight.
Next day add 250 uL 20% SDS, and incubate at 60C for at least 30 minutes, or
until lysis is complete, whichever is longer  Add more SDS if required.  Add
1/3 vol (1.3 mL) 5M NaCl.
Add an equal volume (5 mL) of chloroform:iso-amyl alcohol (CIAA) (24:1), and
hold at room temperature for 30 minutes with intermittent mixing.
Centrifuge at 4000 rpm, RT, 20 minutes.  Transfer supernatant to clean corex
tube, add 1 vol CIAA, mix well, and centrifuge as before.
 Transfer 750 uL aliquots of supernatant to eppendorf tubes.  Add 375 uL of
phenol, and 375 uL of CIAA.  Mix well and centrifuge at 12500 rpm, 5
minutes, room temperature.  Transfer supernatant to fresh eppendorf tube,
and repeat extractions until interface is clear.  Repeat one final time.
 Add 1 volume CIAA and centrifuge (12500 rpm, 5 minutes, room temperature),
and repeat.  Transfer to a 2 mL tube, add 1/10 volume 3M NaAc pH 4.8, and 2
volumes 100% EtOH.  Incubate at -20C for two hours or overnight.  Centrifuge
at 12500 rpm, 5 minutes, 4C.  Wash pellet with 70% EtOH, dry, and resuspend
in 100 uL dH2O.  Incubation at 65C for 1 or 2 hours can help dissolution.

TSBYK :     1L media:
½ recipe trypticase soy broth   15 g
½ recipe brain heart infusion   18.5 g
1% yeast extract    10 g
1% hemin solution    10 mL
Autoclave and add
1 uL/mL vitamin K (filter sterilized)  1 mL
1 uL/mL N-Acetyl muramic acid (Sigma A-3007 for B. forsythus only)

Hemin solution:
50 mg hemin and 1.74 g K2HPO4 in 100 mL H2O.
Boil to dissolve, and store at -20C.

Vitamin K solution:                                                  NAM
4 mg water soluble menadione                             100 mg NAM in 10 mL
in 10 mL dH2O    .
Filter sterilize and store at -20C.                Filter sterilize and
store at -20C.

SET buffer:
75 mM NaCl
25 mM EDTA
20 mM Tris, pH 7.5

Glenn Wall-Manning
Dental Research Group                 Standard disclaimers apply
Wellington School of Medicine
New Zealand
P.M. Klabbers wrote in message <35D9AE38.A93A071D at tref.nl>...
>Can somebody recommend an DNA isolation procedure for:
>Pseudomonas aeruginosa
>Bacillus subtilis
>Staphylococcus aurius
>Pim Klabbers

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