Rescuing plasmid from dead E.coli stock.
hroychow at NMSU.EDU
Fri Dec 6 13:17:52 EST 1996
At 10:00 AM 12/5/96 -0800, Emma Macfarlane wrote:
>I remember reading some time ago on this newsgroup about retreiving
>plasmid from dead plates to re-transform into E.coli, but I can't
>find reference to it in the archives. Is it possible to
>do the same thing with frozen stocks? We have an important construct
>in a sickly E.coli strain that is now refusing to grow. I tried
>taking a scraping of cells, boiling it up in water, spinning it down
>and using the supernatant to transform DH5a. This didn't work.
>If anyone has an alternative suggestion I would appreciate hearing it.
>Please reply direct to my email address emma at hancock.microbiology.ubc.ca
>as I am not a regular subscriber to the newsgroup.
>Thanks in advance
You could always obtain sufficient amount of plasmid DNA from 'dead' colonies left on an agar plate to retransform cells. I suggest you do a 'micro' alkaline lysis prep with the scraped cells in a 50 to 100 uL GET/(lysozyme) solution. I would not bother doing any PCI extraction or running any gel to monitor the yield. If you spin the acidified supernatant twice at 15K/15 min, the resulting plasmid is clean enough. Ppt. the DNA as usual and resuspend the pellet in 50uL TE8. Use 1 to 5 uL of that to transform a 100uL aliquote of competent cells.
Dr. Hiranya Sankar Roychowdhury
Plant Genetic Engineering Lab.
New Mexico State University
Las Cruces, NM 88003
Ph. (505) 646-5785
hroychow at nmsu.edu
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