Cloning Fails-- Trouble shoot please

Jayakumar, R R.Jayakumar at roswellpark.org
Wed Nov 9 13:27:47 EST 2005


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.



More information about the Methods mailing list