<div>Dear Dr. Jaiswal,</div> <div> </div> <div>Thank you for your kind suggestions, i will try it.</div> <div> </div> <div>with best regards</div> <div>sincerely yours</div> <div>Manoj oak<BR><BR><B><I>Pankaj Jaiswal <pj37@cornell.edu></I></B> wrote:</div> <BLOCKQUOTE class=replbq style="PADDING-LEFT: 5px; MARGIN-LEFT: 5px; BORDER-LEFT: #1010ff 2px solid">At Gramene we do not provide support for such queries. However in my <BR>personal experience try to digest your DNA sample with appropriate <BR>restriction enzymes (this is the best way compared to PCR) to check if <BR>your insert of correct size is there or not. Another possibility is that <BR>the colonies if present on old plates can develop contamination. Thus <BR>your contaminant is growing much more than your actual clone. So the <BR>best is to transform the cells again with the plasmid prep of the cloned <BR>fragment you might still have and start your cultures from the new
<BR>transformants.<BR><BR>Pankaj<BR><BR><BR>Mohanty, Amitabh wrote:<BR><BR>> Hi Manoj<BR>> 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.<BR>> Thanks<BR>> <BR>> Amitabh Mohanty, Ph.D.<BR>> Cold Spring Harbor Laboratory<BR>> Delbruck Building<BR>> 1, Bungtown Road<BR>> Cold Spring Harbor<BR>> NY11724, USA<BR>> <BR>> <BR>>
<BR>> <BR>> -----Original Message-----<BR>> From: owner-gramene@gramene.org on behalf of manoj oak<BR>> Sent: Tue 8/29/2006 9:10 AM<BR>> To: gramene@gramene.org<BR>> Subject: Plasmid isolation problem<BR>> <BR>> Dear Grains,<BR>> <BR>> 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. <BR>> <BR>> Please let me know what is going wrong in my case. <BR>> <BR>> I appreciate your help in this regards.<BR>> <BR>> with best regards<BR>> sincerely yours<BR>> <BR>> <BR>> <BR>> If you judge people, you have no time to love them. -- Mother Teresa<BR>> <BR>> Manoj D Oak Ph. D.<BR>> Scientist <BR>>
Genetics Department<BR>> Plant Science Division <BR>> Agharkar Research Institute<BR>> Agarkar Road <BR>> Pune 411 004 <BR>> INDIA <BR>> Phone +91-20-25653680 (Off) <BR>> E-mail: manojoak@yahoo.com<BR>> <BR>> ---------------------------------<BR>> Stay in the know. Pulse on the new Yahoo.com. Check it out. <BR>> <BR><BR><BR>-- <BR>Pankaj Jaiswal<BR>G-15, Bradfield Hall<BR>Dept. of Plant Breeding and Genetics<BR>Cornell University<BR>Ithaca, NY-14853, USA<BR><BR>Ph. +1-607-255-3103 / 4199<BR>fax: +1-607-255-6683<BR><BR></BLOCKQUOTE><BR><p> 
<hr size=1>Do you Yahoo!?<br> Everyone is raving about the <a href="http://us.rd.yahoo.com/evt=42297/*http://advision.webevents.yahoo.com/mailbeta"> all-new Yahoo! Mail.</a>