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Problematic ligation


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#1 HyperD

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Posted 12 August 2004 - 12:55 PM

Hi ppl,
I have a problem with a ligation. I have to ligate a 1.4 kb insert in a plasmid of 14.4 kb. I have been working on it for quite long time now, it's the final step of my cloning process. The 1.4 kb insert is a XhoI digested product, and the backbone of is opened with SalI and after that processed with SAP.
Well, I tried everything and checked all the possible problems. Could the disproportion between the ligation fragments be a problem? Now I use a ligase from Roche, and leave the ligation r[code=auto:0]eaction for 2 h on 16 C. After that I did the transformation with heat shock or with electroporation but none of the colonies grew up. Well... anybody has a suggestion where the problem could be? Thanks

#2 wirly

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Posted 12 August 2004 - 04:23 PM

Since you "tried everything and checked all the possible problems" I don't think I have much to offer. ;)

I've had problems in the past when I was using a larger vector (only 9kb though). I can't say it was the just the size since it is the only "large" vector I've used. Everyone had problems with it and we always chalked it up to size.

2h at 16c seems short. If I'm ging to do a 1-2 hr ligation I do it at RT, if I do it at 16c then it's over night.

I'm not sure all the conditions you've manipulated but I'd go with some extreme vector to insert ratios if you have not already. 1:1, 1:5, 1:50 using 200-500 ng of vector. I mention using more vector because the usual 50-100 ng of vetor applies to vectors 1/3 the size on average. To have the same number of colony forming units you need at least 3X the weight of vector.




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