Originally Posted by harr0750
I am trying to clone a 1.8 kb insert into a 6 kb expression vector. I PCR amplified my target gene adding EcoRI and XbaI sites and digested, ligated and transformed into XL-10 gold competent cells. I get many clones on my plates (with few colonies on my negative control plates) and have run into major problems screening positive clones. When I perform a restriction enzyme digest using EcoRI and XbaI, or a single digest, all of the clones I screen have the same digest profile! (and not at the right size). The uncut, single cut, and double digest (to cut out potential insert) all result in the same banding pattern (~6 kb and ~3 kb). I have tried different restriction enzymes and have tried to select positive clones by PCR, but have had no luck. Any ideas about what could be going on would be greatly appreciated!
I have a suspicion. XbaI is a dam methylation sensitive enzyme. This means the genotype of the E. coli from which you've prepped your vector must be dam/dcm- otherwise XbaI will not cut.
What this means is that the only enzyme which is digesting your vector at all is EcoRI. Since you are getting a ~3 kb band, my guess is that your digested PCR products are annealing at the XbaI site end-to-end, and then ligating as concatamers with EcoRI into your EcoRI digested vector. The insert in this case would be 3.6 kb. Is the "~3kb" band you see actually slightly higher than the marker? Between 3 and 4? This also explains why your single cut EcoRI results in 2 bands. As for the uncut having multiple bands - uncut plasmids always have multiple bands, so the "3 kb" band may just be the supercoiled plasmid. The single digest with XbaI should actually fail and look just like uncut plasmid. However "true" cuts of plasmid will be brighter than the supercoiled band, so the "Uncut" and "single xbaI" should look slightly different from the "EcoRI" and "EcoRI/XbaI".
Switch competent cells and transform your vector into a competent cell line which is dam/dcm neg, do a new plasmid prep of your vector, and do the digestion again. There should be no need to redigest the PCR product, though you may want to do all the digestion again.
There should be no need to transform your ligation product
into dam/dcm(-) E. coli, unless of course you plan to use XbaI to screen your clones, or some other application.di