still more about cloning PCR products
Posted 12 August 2004 - 04:40 AM
I know that cloning PCR products with RE sites in at the end of the primers has been discussed earlier. Anyway, I have a puzziling problem, and I would appreciate all comments.
I have now tried to clone a 200 bp PCR-product with SacI and Not I sites into a vector opened with the same enzymes. I have made all digestions overnight, digested both enzymes in their own optimal buffers, and I have 6 extra flanking bases in the primers. The vector is opened quite nicely ( I can see a small stuffer fragment removed on the gel), and I have gel-purified it. As said earlier, the digestion of insert is quite impossible to monitor.
After transformation I got less that one hundred colonies and roughly same amount of colonies on the control plate. All the clones seem to contain the parental plasmid (with original stuffer fragment).
Does anybody else have (bad) experiences with SacI and NotI in digesting PCR-products? I´m using enzymes from Fermentas.
Posted 12 August 2004 - 06:01 AM
I think you can never be sure that both enzymes cut your vector to 100 %.
Did you try to cut your vector first with SacI and in another experiment to cut it first with NotI and run a gel? There you could perhaps see if one enzyme doesn't cut properly.
But even if the single digestions work, there could be a problem if the second enzyme has to cut close to the end of your single-cut vector. (But if you see your stuffer fragment in the gel I guess that's most probably not the case.)
Did you use an alcaline phosphatase (like CIP)? This could also help preventing your vector from religation.
I'm also using enzymes from Fermentas but digestion usually works fine.
Posted 12 August 2004 - 07:29 AM
Posted 12 August 2004 - 08:19 PM
For certain restriction enzymes (I think Cla1 is an eg.), if you add more than two extra bases, the efficiency of digestion goes down! Check in the NEB catalogue to see how many extra bases will give you the desired efficiency for Not1 and Sac1. Also, if "overnight" means 15 hrs, for smaller fragments that may not be adequate. I generally do atleast 20 hrs of digestion for a PCR product.
Hope that helps.
Posted 18 August 2004 - 05:20 AM
all depends on what u are looking 4....
i agree with the use of PCR designed cloning vectors such as TA ones. the thing is if u need to clone in some particular vector, u should keep on working on it (u ve got nice advices).
BUT, if it is just about cloning PCR pdcts, go 4 a TA vector if u have used a not proof reading Taq pol, and TOPOcloning system (Invitrogen), if it was a proof reading Taq pol (blunt ends). The TOPO system uses a topoisomerase instead ligase, it s a 5 min reaction and very efficient in my experience.
Then, u can cut your fragment from the cloning vector and transfer it to the one u need.
Posted 18 August 2004 - 08:22 PM
Not I is a very difficult one to digested when it close to the end of DNA fragments. check this website, http://content.labve...e_of_Oligos.pdf
and !!! how many oligs it need for a >90% digestion??
u'd better try TA clone. even if u used the proof reading Taq pol(blunt band), just adding A-tail, that's much easier than...NotI digestion.
Posted 19 August 2004 - 04:40 PM
Sometime we have custom sequence synthesized by them (300-600 bp any sequence), quite convenient but sometime you need to wait. Apparently they're doing good business that is over their workload.