Plasmid isolation problem

[manoj oak]

Dear Dr. Jaiswal,
   
  Thank you for your kind suggestions, i will try it.
   
  with best regards
  sincerely yours
  Manoj oak

Pankaj Jaiswal <pj37 at cornell.edu> wrote:
  At Gramene we do not provide support for such queries. However in my 
personal experience try to digest your DNA sample with appropriate 
restriction enzymes (this is the best way compared to PCR) to check if 
your insert of correct size is there or not. Another possibility is that 
the colonies if present on old plates can develop contamination. Thus 
your contaminant is growing much more than your actual clone. So the 
best is to transform the cells again with the plasmid prep of the cloned 
fragment you might still have and start your cultures from the new 
transformants.

Pankaj


Mohanty, Amitabh wrote:

> Hi Manoj
> Getting false positive colonies in colony PCR is very common. One indicator of right colony is the strength of the PCR bands. Were your bands very strong? Secondly, its not uncommon to loose plasmids during prolonged large scale cultures. Its a good idea to not to let the big cultures grow more than 12-15 hours. I have no idea how many Kb fragment you're going to sequence but often you get enough plasmid to do 3-4 sequencing reactions from a miniprep using some of the commercil miniprep kits such as QIAGEN ot Promega. You may try this as well. I'd also recommend to digest the plasmids isolated from the colony PCR positive clones befor you proceed to do large prep.
> Thanks
> 
> Amitabh Mohanty, Ph.D.
> Cold Spring Harbor Laboratory
> Delbruck Building
> 1, Bungtown Road
> Cold Spring Harbor
> NY11724, USA
> 
> 
> 
> 
> -----Original Message-----
> From: owner-gramene at gramene.org on behalf of manoj oak
> Sent: Tue 8/29/2006 9:10 AM
> To: gramene at gramene.org
> Subject: Plasmid isolation problem
> 
> Dear Grains,
> 
> I cloned LMW glutenin genes (of wheat) in pMOS-T blue and also pGEM-T cloning vectors, I confirmed positive clones using colony PCR, I used vector primers and also specific LMW glutenin primers. I got expected size amplicon. Then I incubated positive clones to isolate plasmids (alkaly lysis method) in large ammount for sequencing. But i am not getting the plasmids. 
> 
> Please let me know what is going wrong in my case. 
> 
> I appreciate your help in this regards.
> 
> with best regards
> sincerely yours
> 
> 
> 
> If you judge people, you have no time to love them. -- Mother Teresa
> 
> Manoj D Oak Ph. D.
> Scientist 
> Genetics Department
> Plant Science Division 
> Agharkar Research Institute
> Agarkar Road 
> Pune 411 004 
> INDIA 
> Phone +91-20-25653680 (Off) 
> E-mail: manojoak at yahoo.com
> 
> ---------------------------------
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-- 
Pankaj Jaiswal
G-15, Bradfield Hall
Dept. of Plant Breeding and Genetics
Cornell University
Ithaca, NY-14853, USA

Ph. +1-607-255-3103 / 4199
fax: +1-607-255-6683



 		
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