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Ligating Incompatible DNA fragments


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3 replies to this topic

#1 sayem769

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Posted 28 June 2011 - 09:54 PM

Hi,
I am trying to ligate my gene into a new vector. In order to cut my gene out of the current plasmid I have used SmaI and SacI restriction endonuclease.
The vector i want to insert my gene in is designed to be cut with BsmBI. So my question is, is there a way for me to cut my insert with SmaI and SecI and then ligate it with the vector which has been cut with BsmBI?
I know those enzymes are incompatible. If there is a way, could someone please tell me how?

Thank you in advance.

#2 bob1

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Posted 29 June 2011 - 01:27 AM

How about blunting (nibbling off the overhang - can't recall the enzyme at the moment), overhang filling, or ligation of adapters onto your cut ends?

#3 GNANA

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Posted 29 June 2011 - 01:54 AM

Why not you amplify your insert from the current plasmid with both primers containing Bsmb1 overhang? may be this will make your job easier (assuming that you dnt have this Restriction site anywhere within the insert)..

Edited by GNANA, 29 June 2011 - 01:55 AM.

I would prefer being perfectionist rather than a passionist in Research.

I always had an alternate hypothesis....

#4 Evanescence

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Posted 29 June 2011 - 05:24 AM

why dont you design a set of primers to amplify your gene...and yeah the primers of course having BsmBI recognition restriction site...pcr, digest and ligate...




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