Hello everybody,
I have an expression vector about 4600 bp. I cloned my insert in it and made colony PCR. I got very good amplification, so I isolated the plasmids and digested if everything is okey. It should give one band around 360 bp, but now I have 3 bands which are 250,360 and 650. Any idea?

Doing a colony PCR is quick and easy but the temptation is to choose the colony that gives the brightest PCR band. Unfortunately, this is likely to be a clone that has more than one insert. If you look at your gel picture again count how many clones were weakly positive, how many appeared to have about twice the signal and three times the signal etc. You would expect > 50% of the clones with any signal to have the weak signal (monomeric insert) and then fewer and fewer to have the stronger signals- these will have concatomerised inserts.



You can prevent this happening by dephosphorylating the plasmid prior to ligation and using a much reduced amount of insert. As a rule of thumb, a molecular ratio of 1:1 is good so you should use no more than 1/12 as much insert as you use of the vector (eg. 500ng vector-dephosphorylated + 40ng insert).

Among positives, only 1 gave weak signal, the others were very bright. You're right that it could have more than one insert. I have thought it before, but the bands after digestion weren't suitable for this situation too. I tried to think all the alternatives, but have no result yet. Today, I'm digesting other vectors which have my inserts also. I hope one works well. If all gives the same result, I will have the biggest problem I suppose.

I tested both. With enzymes only my inserts have and vectors enzymes, also vector and insert's enzymes.

My insert is PCR product and it has HindIII and XbaI sites on its ends.
I have checked 7 colonies and 3 gave amplified fragment at 360 bp. I have cut 2 of them and they have given same band pattern (vector+250+360+650bp), fortunately the left one gave correct bands after RE digestion and I can keep on cloning the other fragment.
I suppose my insert + a fragment from vector which is cut for cloning were cloned into the vectors, therefore I observed an unwanted band pattern. There is no explanation besides it, cos I'm sure that it hasn't got more than one insert.

If I understand correctly, the final colony you checked was as you had hoped and gave just the 4600+360bp bands after RE digestion. Excellent.

Since you only had 7 colonies to begin with after transforming the bugs with the ligation reaction, I'd guess that the H3 and XbaI sites at the ends of the PCR product were very inefficiently cleaved. I have an old New England Biolabs catalogue which I use to check how well the various enzymes cut at the end of molecules and it suggests that H3 may be a problem but that Xba I should be OK. Here's a link to the same info from the NEB website:

http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/cleavage_olignucleotides.asp


Although I used to do a lot of PCR cloning this way I found using a proof reading polymerase and the TOPO-Blunt kit from Invitrogen to be such a lot more efficient. There are now quite a few other companies with similar products (Stratagene and Fermentas) so you should be able to find one that has RE cloning sites that fit your purpose. It also makes it more cheap & convenient to sequence using the T3, T7, M13 etc primers before going on to the next cloning steps.


PM

Thanks for all your help Parvoman. You understood correctly, I had the colony I want.
The site you sent the link is very useful, thanks again.
I'm also using a proof reading polymerase for cloning. My vector pJET1 can be easily sequenced by T7 sequencing primer, but my mammalian expression vector has unique sequencing primers, so I have to find another solution.