problem with ligation of big fragments - (Aug/21/2007 )
Hi everybody! : I´m trying to ligate an insert of 2257 pb with a plasmid of 10 kb. The original plasmid has 13kb. I cut it with SphI and NheI to release a fragment of the same size that the fragment I want to insert. I cloned my insert (product of PCR) in a pGem-T vector and release it with SphI and NheI too. Then I purified by gel the insert (2257pb) and the lineal plasmid (10kb), both cutting with SphI and NheI. I´ve tried the ligation with different relation 1:1, 1:2 1:3 and 1:5 of both fragments, but when I did the screening of the colonies (about 100 by plate) all were negative. The control with only the lineal plamid had some colonies, so is probably that they had plasmid that the enzymes didn´t cut. I’ve tried again cutting the plasmid with more enzyme and for more time. The next transformation gave colonies in the control plate of insert and lineal plasmid. So the positive colonies for the insert were those that carries the pGem-T with the insert. So I’ve desfosforilated the purificated insert to avoid the religation with pGem-T. (the insert and pGem-t have similar size). In the next transformation I had colonies only in the control lineal plasmid but not in the control insert plate and I didn´t have positive colonies. So my question is: Is it possible to ligate these fragments ?. I’ve working with Max efficient DH5 alpha for the transformation and I’m trying to obtain the purest fragments for the ligation.
Thanks in advance
Something that really help me is to mix vector and insert with the water and incubate as 45 o 50°C for 5 minutes, then let them warm at room temperature and then add buffer and ligase. Believe me, it really has increased my efficient ligation!
pGEMT is 3 kb and your insert is 2.25 kb, I think you can perfectly separate them in a 5-7 % agarosa gel, if not, cut with SphI, NheI and with a enzyme that cut in the middle of pGEMT and that don’t cut your insert, this way, you’ll have a 2.25band (your insert) and 2 smaller bands (pGEMT) so you can purify just your band.
hope this help!!!
I certainly agree with aztecan princess recommendation. Cut the pGEMT backbone is to smaller fragments by a third enzyme. This will help remove the desired insert. If anything it is the vector that needs to be dephosphorylated.
Just a question, the insert:vector ratios you are quoting, are the molar ratios? Mass ratio and volume ratios do not work well when the fragment sizes are so dissimilar.
What transformation method are you using? Are you using home made or company made competent cells? While it is stiill possible to do ligations using home made cells at plasmids this size, if things continue to fail, you might consider using company made cell which are more competent.
And for further assurance, Yes. this is certainly doable. You can do this. A labmate of mind has conducted a ligation between a 190kb insert into a 14kb vector.
Thanks for your advices. I´ve worked with molar ratios at the ligation and Max Efficient DH5aplha of Invitrogen for the transformation (termic shock at 42ºC 45 sec). In the next ligation I will try with the incubation at 50ºC for 5 min before adding the ligase and buffer. Also I purified the insert from pGemt: first I cut the pGemT with DraI (so it divided in 3 fragments) and then I cut it with SphI and NheI to release the fragment of 2257 pb.