Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > Protocols and Methods Forum
Register Search Today's Posts Mark Forums Read

Protocols and Methods Forum Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


Cloning Fails-- Trouble shoot please

Cloning Fails-- Trouble shoot please - Protocols and Methods Forum

Cloning Fails-- Trouble shoot please - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 11-09-2005, 06:27 PM
Jayakumar, R
Guest
 
Posts: n/a
Default Cloning Fails-- Trouble shoot please



That is what they call self-ligation. It is very easy to check whether the plasmid is self ligated or not. Just run the "ligated" construct along with your original plasmid on a gel. The last band (the supercoiled form) and I believe all the other forms will run more slower in the ligated vector since they are larger in size. There was a publication in 1978 or 1977 in the journal Plasmid which describes this phenomenon.
Gonzalez, Jr. J.M., H.T. Dulmage and B.C. Carlton. 1981. Correlation between specific plasmids and -endotoxin production in Bacillus thuringiensis. Plasmid 5: 351-365.
The technique described in this paper helps you to screen 100s of clones pretty fast to check for ligated vector within a day. I have a more simplified version of the procedure if you need. I have effectively used this procedure even for checking for insert concatamers in the plasmid. Larger the insert more slower the different forms of the plasmid will run with relation to the original plasmid.

Best of luck
Jayakumar



This email message may contain legally privileged and/or confidential information. If you are not the intended recipient(s), or the employee or agent responsible for the delivery of this message to the intended recipient(s), you are hereby notified that any disclosure, copying, distribution, or use of this email message is prohibited. If you have received this message in error, please notify the sender immediately by e-mail and delete this email message from your computer. Thank you.

Reply With Quote
Reply

Tags
cloning , fails , shoot , trouble


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
Trouble with Cloning into Lentiviral Vector Gam8Doc Molecular Cloning Forum 3 06-11-2013 02:17 PM
help on difficult cloning Senkovich, Olga Protocols and Methods Forum 1 05-05-2007 12:14 AM
BioInfoMan:reduce your working time on molecular cloning by 80% gaxl Bioinformatics 2 08-15-2006 12:31 AM
FW: Cloning Fails-- Trouble shoot please Deanne Bell Protocols and Methods Forum 1 11-11-2005 01:43 PM
Cloning Fails-- Trouble shoot please vijaykr Protocols and Methods Forum 7 11-09-2005 04:27 PM


All times are GMT. The time now is 11:21 PM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.11321 seconds with 16 queries