clonning problem (recombined by-products) - (Oct/10/2011 )

I'm trying to clone a 2kb fragment into a 10 kb vector. I get a a lot of colonies on my plate but most of them are neither the right clone nor the empty vector a new construct which does not even contain the two restriction sites used for this cloning ! It seems that the construct recombines inside the bacteria. does any body know some trick to minimize this? other than using rec(-) cells which I'm not sure how much it will help! Any special consideration in my ligation set-up, transformation or cultures?

Essentially all cloning strains are recA-. What are you cloning into? Does your plasmid have repeats?

Well the story is that I have flanked the expression cassette by two "insulator sequences" (exactly the same and each 400 bp) and now I am doing the last step which is inserting the CDS inside the cassette. without these insulator sequences I had no problem with the cloning but now it's become a nightmare ... people have done this before and use the resulting backbone for their routine cloning so there must be a way to do it !!!

Two 400 base direct repeats are very likely to cause recombination in a strain which is recA+. This will remove whatever is between the repeats and one of the repeats. If you have a choice, make the two 400 bp sequences different.
Again: what strain are you cloning into?

I'm cloning into dH5alpha cells!

Well, I agree that the argument "other people have done it before and it worked" is compelling, its hard to argue that its not working now and its better to get it working whatever way you can rather than trying to work backwards.

Were the insulator sequences that other people used the same as yours? Same plasmid backbone? Same insert? It is possible that any combination of these could be triggering some form of recombination. Its possible that just the ligation of that particular insert triggers the recombination. I would try to obtain a more genetically debilitated competent cell line such as XL-10 gold or StBL2 cells that tend to handle unstable plasmids better. You could also try growing the tranformation plates at a lower temperature like 30 degrees or room temp. The colonies may take 2 days to grow, but the plasmid may be more stable.

Lastly, is it possible to change your cloning strategy? Could you clone one insulator sequence into your vector, then the insert, then the second insulator? I know that this may seem more cumbersome, but what is even MORE cumbersome is trying the same failed cloning over and over.

Best of Luck.

just a question out of curiosity
...what should be the use of these insulators?
...do they need to have exactly the same sequence?

using STBL or SURE cells is a good idea ...but since your repeats are that long the question is if they can prevent the recombination? ...lower temp as allynspear mentioned is also a good idea!
but in general direct repeats tend to be very unstable in E. coli ...so i would think about chaning your strategy.

Regards,
p

to allynspear and pDNA,

These insulator sequences are used to block the position effect on the expression level when the construct is inserted into the fly genome. A similar construct which has done this effectively used the same sequence on both sides and now I'm following that strategy.
I totally agree with you guys that I may need to follow a different cloning strategy such as cloning one insulator, then the CDS and finally the second insulator. but before that I would like to try a few other E.coli strains as well as growing at a lower temperature; also I'm doing my ligation this time at 14'C overnight instead of RT 2 h, and 20ul reaction instead of 10 ul; I don't know if these could also make a difference! we'll see ...

I got my clone finally! Yay .... DH10B cells did it for me