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#1 janani



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Posted 15 March 2011 - 02:32 AM

hi all,
i am having a problem with cloning a 13.2kb plasmid that has 7kb insert and 6.2kb vector. I get the ligation done and it does work cos i check it on the gel and it gives a smeared but single ligated band. but when i did chemical transformation, i got no colonies whatsoever. After a lot of trials i switched over to electroporation and i did it with dh5 alpha and xl-10 gold. I got colonies and got plasmids from them always, but restriction to check for insert release gave no more than 9kb or 10kb linear plasmid, neither vector nor my insert is getting inside fully or properly. but how can random parts of them get cut out?
restriction with xho1 and not1 (the sites that i used to ligate) in the different electroporation trials gave:
trial 1: a 5kb band and a 4kb band. total of nine kb.
trials 2 to 7: random bands with any restriction enzyme, not matching the size of my vector or insert.
my vector is fine cos i used it to clone two more inserts. My insert is pcr purified fresh. Where am i going wrong? plss help.

#2 bob1


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Posted 15 March 2011 - 04:33 PM

How do you extract your plasmids? If you use any method that involves alkaline lysis (this includes most kits), then the presence of small amounts of medium during the extraction proceedure can cause restriction enzymes to not work properly.

#3 janani



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Posted 18 March 2011 - 08:13 PM

yes, my kit includes alkaline lysis. but i tried manual method as well. i am doing the same extraction for other plasmids and it seems to work fine :(

Edited by janani, 18 March 2011 - 08:14 PM.

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