Cloning PCR product - Any ideas? (Jun/03/2006 )

I do PCR to the gene (2.3 kb) out from a plasmid and create different restriction enzyme sites at the ends (NHeI and EcoRI-not suppose to be compatible). I purify the PCR product on column using a kit from Promega. I digest the PCR product and the plasmid YFP-N1 from Clontech (4.7 kb) with NHeI and EcoRI and gel purify again with Promega. I do elutions with 15 microl elution buffer and get a nice concentrations 30ng/microl. I set up a ligation in vector:insert ratio 1:3 with fast ligation kit from Epicentre (total volume for ligation 15microl). I transform 2 microl of ligation into 100 microl of DH5alpha competent and plate all the 100 microl. I get a lot of colonies-400-500. Just to be sure I pickup 20 colonies and do PCR with primers containing the NHeI and EcoRI restr.sites- and get nothing.
Someone has any idea? Thanks.
Sem

Perhaps one of your enzymes is not cutting? Then your vector would only be linearized with a single enzyme, and would ligate back together quite readily.

How did you prepare your vector? Did you gel purify it after digestion? What happens if you do a mock ligation, including linearized vector-only and no insert? If you get hundreds of colonies from that, your vector is inadequately prepared.

How many irrelevant base pairs did you include on the 5' end of the restriction sites engineered into the primers?

you say you're cutting your plasmid with 2 enzymes...
But how long is the fragment generated between these sites (i'm not talking about the vector)

If it's relative small better is to avoid gel purif.

Hi, HomeBrew and fred 33

Before I did a double digestion, I checked every enzyme alone- they worked. I have 3 irrelevant base pairs on 5' before the restriction site. After digestion I gel purify the plasmid (I cut it from the gel) and the fragment generated after digestion (inside the MCS of the vector) is 20bp long.
Dear fred 33, what do you mean saying