Plasmid isolation problem

[Mohanty, Amitabh]

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
   


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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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