How to get rid the restriction sites in a plasmid ? - (Feb/15/2008 )
Hi all
i am trying to get rid the restriction site in a plasmid with out doing site-directed mutagenesis.
i just digest with particular enzyme, transform to E.coli and select survivors, grow up all survivors, and repeating. i repeated several time but still i am unable to get rid that site. if some one has idea on it please share with me ..
thank you
If you are unconcerned about disrupting a reading frame, then you can digest with your enzyme, fill in with klenow, and then religate.
I guess it all depends on where the site is. If it isn't in a coding or essential area you could try doing a digest (assuming the restriction site leaves overhangs), fill it in with klenow and then blunt end ligate it. Do you have a map of this plasmid anywhere and can you say which site it is? I could be more specific if I know more about your plasmid.
* Edited *
Dang, Smu2 beat me to it as I was typing!
i am trying to get rid the restriction site in a plasmid with out doing site-directed mutagenesis.
i just digest with particular enzyme, transform to E.coli and select survivors, grow up all survivors, and repeating. i repeated several time but still i am unable to get rid that site. if some one has idea on it please share with me ..
thank you
How would this help getting rid of that site? The colony's can only grow if they contain a circular plasmid, so you will only grow plasmids that are not cut if I undertand you correctly.
i am trying to get rid the restriction site in a plasmid with out doing site-directed mutagenesis.
i just digest with particular enzyme, transform to E.coli and select survivors, grow up all survivors, and repeating. i repeated several time but still i am unable to get rid that site. if some one has idea on it please share with me ..
thank you
How would this help getting rid of that site? The colony's can only grow if they contain a circular plasmid, so you will only grow plasmids that are not cut if I undertand you correctly.
After blunt ending the restriction site, do a ligation. This will give you recircularised plasmid.
If indeed you include the blunting step, but that wasn't in the post so that's why I asked and mentionend "If I understand you correctly". (you never know wether or not people post all information on their experiments).