TOL Plasmid Prep - protocol

K.G. Korth kkorth at kcc.com
Thu Mar 28 14:58:19 EST 1996


A few months ago I answered a posting on this newsgroup related
to recovering TOL plasmids from Pseudomonas spp. and received a
fair amount of response asking for more details on the method.
The method I used (I say used, because this was several years
ago) was a modification of the Anderson and McKay method for
isolating large plasmids from Lactic Streptococci (Applied &
Environmental Microbiology. 1983. 46:549-552).  I have received
numerous requests since the posting asking what the modifications
are.  Therefore, I am putting the entire method here, on this
posting.  I wish anyone luck while working with TOL plasmids.
They are easily the toughest plasmids I've ever worked with.  I
tried at least a dozen different methods for isolating TOL and
found all to be woefully inadequate, due mainly to their size
(ca. 117 kb) and their low copy number (often only one per cell).
There may be something out there better, as I have been out of
the TOL arena for years, but this worked well for me and I
regularly recovered milligram amounts of the plasmid, suitable
for molecular study.

Here are the modifications on the Anderson and McKay prep for
recovering TOL plasmids.

I always used the 50 ml tube (oakridge tube) volume, though the
250 bottle volume has worked well for some of my co-workers.

Grow up the cells overnight with shaking in LB containing 10 mM
4-methyl benzyl alcohol and 10 mM m-toluate (you will have to do
some major pH work with heating after adding the m-toluate, if you
buy it in the form of m-toluic acid, to get the pH back up to
physiological level).  Both of these additions can be added prior
to autoclaving.  The m-toluate will flocculate out a bit, but the
media still works.  Chill your culture on ice then pellet it in
250 ml bottles (ca. 100-200 ml) before resuspending in 5 ml of
the sucrose solution and transferring it to oakridge tubes for
the rest of the prep.  Get P.putida mt-2 ATCC 33015.  It
contains the archetype TOL plasmid (good positive control), but don't 
transfer it on benzoate agar (as ATCC suggests), rather transfer it on 
agar made from the LB modification listed above.

Amounts are given in two volumes, the first representing 100 ml
pelleted in a 50 ml oakridge-style tube, and the second
representing 500 ml pelleted in a 250 ml bottle.

1.   Resuspend pellet in (6 ml/30 ml) 6.7% sucrose-50 mM Tris-1
     mM EDTA pH 8.0

2.   Warm to 37*C

3.   Add (1.5 ml/ 7.5 ml) lysozyme (10 mg/ml in 25 mM Tris, pH
     8.0)

4.   Incubate for 5 minutes at 37*C

5.   Add (0.75 ml/3.75 ml) 0.25 M EDTA-50 mM Tris pH. 8.0

6.   Add (0.45 ml/2.25 ml) SDS (20% w/v in 50 mM Tris-20mM EDTA
     pH 8.0)

7.   Mix immediately

8.   Incubate for 5 to 10 minutes at 37*C to complete lysis.

9.   Vortex at highest setting for 15 seconds (30 for gram positives) in 
     appropriate tube. (50 ml or less) [note: for many Gram negative     
     bacteria vortexing is not necessary, just inversion mixing. Care    
     should be taken to minimize genomic shearing following lysis]

10.  Add (0.48 ml/2.4 ml) freshly made 3.0 N NaOH.

11.  Mix by gentle intermittent inversion or swirling for 10 minutes.

12.  Add (0.78 ml / 3.9 ml) 2.0 M Tris-Cl. pH 7.0

13.  Continue gentle mixing for 3 minutes.

14.  Add (1.14 ml/ 5.7 ml) 5.0 M NaCl

15.  Add (12 ml/ 55.8 ml) phenol saturated with 3% NaCl: mix
     thoroughly. NO VORTEXING. (repeat if interphase is
     excessive)

16.  Centrifuge at ca. 5000 rpm for 10 minutes

17.  Remove upper phase (avoid interphase) and extract with (12
     ml/55.8 ml) chloroform-isoamyl alcohol 24:1. (once or
     twice).

18.  Remove upper phase and precipitate with one volume of isopropanol.

19.  Incubate at 0°C >60 min.

20.  Centrifuge 8000-10,000 rpm for 20 min.

21.  Remove excess isopropanol by aspiration. Do not disturb the
     pellet.

(At this point you may want to wash your pellet in 75% ethanol a
couple of times recentrifuging after each wash)

22. Dry pellet under vacuum. (do not over-dry)

23. Resuspend pellet in (250 ul, 1.2 ml) TE pH 7.5

After completely dissolved, do a CsCl (1.5 g/ml, 45k RPM for 16 hours, 
vertical rotor) ultracentrifugation to remove the massive amounts of RNA 
typically recovered and any cellular debris.  Save all bands from the 
CsCl (one may be genomic carryover). The three plasmids forms should be 
apparent.

Do a restriction digest on all CsCl recovered bands (XhoI is best) to 
confirm plasmid DNA.


Hopefully those requesting this information will find this useful.


Cheers!

-- 
Kevin G. Korth
KCC Microbiology Research Laboratories
Neenah, WI 54956
E-mail: kkorth at kcc.com	

The ideas expressed here are my own and not
necessarily that of my employer.





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