cloning 11 kb fragment - (Dec/29/2005 )
Hello
I'm trying to subclone a long fragment, 11 kb, from a XL-TOPO vector in to pBuescript.
I release the fragment by a SacI-NotI digestion, then it is separated by electrophoresis and purifyied from agarose. pBluescript is treated in the same way. Vector and fragment were checked on agarose gels for integrity and concentration. Both were ligated using several vector/insert ratios (1:1, 1:5 and 1:10). Ligation were carry out for all night at 14ºC in a 20 uL reaction volume, inactivated at 65ºC and 5 uL were used to transform chemically competent DH5 cells. After palted onto LB-carbenicillin plates amended with Xgal & IPTG, I got blue and white colonies. Plasmids obtained from white colonies doest'n contain the insert (I have analyzed about 20 colonies from each ligation). It seems a small fragment was inserted (but I dont know how it is there!!).
I have been looking for a similar situation in literature and it seems pUC are used to cle big framents (XL-TOPO vector has a pUC ORI)
Any kind of comments, hints and help suggestions will be very wellcome
Gaston
I'm trying to subclone a long fragment, 11 kb, from a XL-TOPO vector in to pBuescript.
I release the fragment by a SacI-NotI digestion, then it is separated by electrophoresis and purifyied from agarose. pBluescript is treated in the same way. Vector and fragment were checked on agarose gels for integrity and concentration. Both were ligated using several vector/insert ratios (1:1, 1:5 and 1:10). Ligation were carry out for all night at 14ºC in a 20 uL reaction volume, inactivated at 65ºC and 5 uL were used to transform chemically competent DH5 cells. After palted onto LB-carbenicillin plates amended with Xgal & IPTG, I got blue and white colonies. Plasmids obtained from white colonies doest'n contain the insert (I have analyzed about 20 colonies from each ligation). It seems a small fragment was inserted (but I dont know how it is there!!).
I have been looking for a similar situation in literature and it seems pUC are used to cle big framents (XL-TOPO vector has a pUC ORI)
Any kind of comments, hints and help suggestions will be very wellcome
Gaston
As far as I know, pBlueScript is better than pUC series to clone a big fragment. Though pUC is okay, but not very efficient, to clone 11kb fragment.
Did you check the negative control? (ligation without insert) And did you find similar proportion of white colonies? LacZ' may be mutated or inactivated. If so, I guess you failed in cloning. Heat shock transformation may not work on the large fragment. Try electroporation
Also I recommend to purify your vector from agarose gel to reduce "possible small fragments" to be inserted into the digested vector.
I did new reactions but now plasmid and insert were purified from agarose. Just 1:5 and 1:10 vector/insert ratios were tested. Now, most of the clones were the expected ones.
Thanks for the suggestions
Gaston