problems with cloning-transformation - (Jun/11/2005 )
Hi! i'm trying to clone a 10 kb insert into a vector (6 kb). Is it possible to do it with chemical competent cells? I tried many times to clone the insert in the vector in different ways, but I never get the right colonies... I wonder if 16 kb are too much.... I heard that electrocompetent cells are better for big plasmids. Is that true?
Thanks a lot!!!
You can try it, I doubt it will hurt, but I doubt it will help either. A 6kb plasmid should be able to take up a 10kb insert without too much trouble.
Have you run an aliquot of the ligation on a get and compared it with the negative control?
I would also double check your cloning/ligation scheme to see if the problem lies there.
First of all, thanks for your reply!
I did not look the ligation on a gel, I will try it.
I tried a lot of different ways to clone the insert. Actually I have this gene cluster (with one gene for Kan resistance) cloned in pUC18. Now I have to transfer the cluster to a staph-E.coli shuttle vector, but I did not manage to do it... Last thing I did was to amplify with PCR the staph ori+ Cm resistance. Tha primers had both a KpnI restriction site + 3 bases upstream(I looked in the neb catalogue, it should be sufficient). Then I isolated the PCR product from a gel (cause I had some unspecific bands). Afterwards, I cutted the plasmid (which has 1 KpnI site) and the PCR product overnight. I zipped the plasmid with CIAP, then I ligated the two with a molar ratio insert vector 3:1, with ca 90 ng total DNA concentration in the ligation reaction (15 ul). Neb says it should work better if conc is betweeen 1-10ng/ul.I performed ligation over night at 14 °C. For transformation, I used Stratagene XL 10-Gold cells. I had apparently a quite good result on plates. The religated zipped plasmid gave ca 5 colonies, whereas the ligation (insert+zipped plasmid) gave me ca 100 colonies. But then I did colony PCR + minipreps, but all the colonies were false positives...
The bad thing is that I can not use Cm for selection, so I can not select for the insert.
Thanks and have a good sunday!!!