LB vs. TB?

malaine at sara.cc.utu.fi malaine at sara.cc.utu.fi
Tue Nov 16 08:48:09 EST 1993


In article <1993Nov16.002307.9371 at alw.nih.gov>, bernard at elsie.nci.nih.gov (Bernard Murray) writes:
> 
> Hello,
> 	Earlier in this thread it was implied that cultures grown in TB are
> unsuitable for mini-preps by alkaline lysis.  So, how do you prepare your
> litre preps from this medium?  Can you use methods based upon glass/resin or
> is CsCl essential?
> 	(Anything to get more plasmid for less effort)
> 				Bernard
> 
> Bernard Murray, Ph.D.

I've used the following protocol to isolate plasmid DNA when grown the bacteria 
in TB medium. The quality of DNA is great, I have directly used it for sequen-
cing. Here it goes:

1.  Grow bacteria in TB for O/N. 
2.  Spin 1.5 ml of the O/N culture at 12400 rpm for 4 min. Discard the
    supernatant.
3.  Resuspend the pellet in 200 ul suspension buffer
4.  Add 400 ul freshly prepared lysis sol, mix the tubes gently and incubate on
    ice for 5 min
5.  Add 250 ul 10 M NH4OAc, mix gently and incubate on ice for 10 min.
6.  Spin at 12400 rpm for 5 min
7.  Transfer the supernatant to a fresh tube, add 0.6x vol isopropanol and
    incubate at RT for 10 min.
8.  Spin at 12400 rpm for 15 min.
9.  Wash pellet with 0.5 ml cold 70% EtOH, spin and dry under vacuum
10. Dissolve the pellet in 200 ul TE
11. Add 83 ul 10 M NH4OAc, mix and incubate on ice for 20 min.
12. Spin at 12400 rpm for 5 min
13. Transfer the supernatant to a fresh tube
14. Add 0.6 ml cold 95% EtOH and incubate at RT for 10 min
15. Spin for 15 min at 12400 rpm and remove supernatant
16. Wash the pellet with cold 70% EtOH, spin and dry under vacuum
17. Dissolve the pellet in 50 ul water

The yield of the DNA is appr. 30-40 ug/ 1.5 ml of TB culture, when
using pBluescript

Suspension buffer:
25 mM Tris-HCl, pH 8
10 mM EDTA

Lysis solution:
20% SDS		125 ul/2.5 ml
2 M NaOH	250 ul/2.5 ml
dH2O		2.13ml/2.5 ml


Hopefully this helps!
Cheers, Marko 

Malaine at polaris.utu.fi
Malaine at sara.cc.utu.fi



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