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Help: incompatible RE site subcloning


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#1 Eric_W

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Posted 15 December 2009 - 11:11 AM

Vector A: 5' NheI, insert, 3' AflII (no other RE sites)
Vector B: 5' NheI, 3' BsiW1 (no other RE sites)

I am trying to subclone the insert in A into B without destroying BsiW1 because BsiW1 will be used later. NheI can be used at 5', but AflII and BsiW1 are not compatible. I don;t want to do PCR because the insert is kind of big.

I am thinking of using 2 oligos to make an adapter which will have AflII overhang and BsiW1 overhang, then do 3-way ligation. but don't the details: e.g. how to design the oligos, annealing conditions....

Is the 3-way ligation a good idea? Any other ideas to do this subcloning? You help is greatly appreciated!

P.S.
AflII: C/TTAAG
BsiW1: C/GTACG

Edited by Eric_W, 15 December 2009 - 11:36 AM.


#2 jangajarn

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Posted 18 March 2010 - 06:43 PM

As far as I know, 3 way ligation could be very complicated. The ratios, DNA quality, proper digests, phosphatase treatment.. too many difficult parameters to control. If I were you I'd serioulsy consider the possibility of PCRing the insert from vector A with a reverse primer having BsiW1 site (see below)

If this is the end of your DNA to be cloned from vector A, 5'.....AGTGAGATCGGAACCTG3'AflII site
design the reverse primer like this: 5'BsiW1 siteCAGGTTCCGATCT....3'




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