Extracting lagre plasmid (~80kb) from Pseudomonas strain

Susanne Kjemtrup skjemtru at email.unc.edu
Thu Jun 11 08:49:34 EST 1998


HI Waiki!
We've had good luck in Pseudomonas syringae strains extracting plasmids
using a miniprep proceedure, modified from the typical alkaline lysis
method. Sometimes it also helps to try to amplify the mega-plasmid with
chloramphenicol first.
1. Spin 1.5 ml culture
2. resuspend pellet into 100 ul solution I + 4 mg/ml lysozyme and 0.1
mg/ml RNAse
3.incubate 10' RT
4. add 200 ul sol II, incubate 10' rt
5. add 30 ul alkaline phenol (0.5 ml phenol, 1 ml NaOH, 0.2M)
6. add 150 ul sol III
7. incubate 20' at -20 C
8.spin 3' rt max speed
9.collect supernatant and extract with phenol, choloroform, IAA
10. Precipitate with 100% etoh
11. wash pellet with 70% etoh
12. Resuspend in 30 to 40 ul TE
To see plasmids on gel, load about 10 ul on a 0.7% TBE agarose gel, run
about 110 V, then run at 15-20 V overnight. You can cut the plasmids
first, then just run the gel as you normally would.
Solutions I, II and III are the typical solutions used for alkaline lysis.
Hope this helps!
Susanne
 
In article <357e2d5a.4648592 at news.caps.maine.edu>, io30634 at maine.maine.edu
wrote:

> Hi,
> Does any have a working method (besides CsCl) for extracting large
> plasmid DNA from Pseudomonas strain? If you do, please let me know
> where I can find it. I tried alkaline lysis and the boiling method
> both are not working. Thanks in advance.
> Waiki



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