gateway cloning: is BP-cloning "reversible" by LR-reaction? - (Aug/25/2011 )
I have a question concerning Invitrogen's GATEWAY cloning. I received an entry-vector prepared from pDONR207 with a PCR-amplified gene. Now I want to get the empty pDONR207 vector to use it for other genes. As I understand the gateway technology, it should be possible to perform a LR reaction with the entry vector and an empty destination vector and the products should be the expression clone and the empty pDONR vector which I then should transform to DB3.1 cells and select on gentamycine/chloramphenicol media. I tried this now three times but did not receive what I want.
Is my idea totally wrong or is there any other suggestion that you can give? It would be very nice if you could help me as this would save us the money to by the vector. We have other pDONRs as well here but cannot use them for a set of dest vectors as they have all the kanamycin resistance.
I once tried to make an empty destination vector by performing a BP reaction with the empty pDONR207 vector and then selecting on gentamycin. At first, all clones contained both the bands from the destination vector with the insert in, and the empty pDONR. Apparently, in the BP reaction, there is an intermediate plasmid formed which is a fusion of the two plasmids, and this is what I got in the end, as it contained the ccdB gene and the gentamycine resistance gene. To avoid getting this, I digested the reaction with an enzyme that cuts in the gene that was cloned in the destination vector. This solved the problem and I could isolate the empty destination vector. So I would advise you to check for an enzyme that cuts in the gene you have in the pDONR but not in the Gateway cassette you want to transfer into it. After doing the LR reaction (but before adding the proteinase K), just add some of the enyzme and its buffer, incubate for an hour, treat with proteinase K and transform (as always, in DB3.1).
As for the incompatibility between pDONR and pDEST vectors with Kana resistance, I solve this in a similar way. In the vectors we have, the Kana resistance gene is not identical in the entry and destination vectors. Apparently, NruI and PvuI cut in the entry vector but not in the destination vector. So when I want to transfer a gene from an entry vector with Kana resistance into a destination vector with Kana resistance, I just do the LR and afterwards (but again before proteinase K treatment), I cut with NruI or PvuI (be sure to check whether these don't cut in your gene of interest that you are trying to transfer), do the proteinase K treatment and transform. This normally works as if there were two different antibiotic resistance genes.
thx for your suggestion!
so far i digested the entry clones from the kana vectors with NruI before i performed the LR reaction and did also gel extraction of the linearised product. but still there seemed to be several side products after LR so that taken it all together digestion, gel purification and selection for the correct expression vectors was always too much work to use it for a high number of clonings. but maybe it will be easier if i do digestion directly after the LR reaction. i do have to check the dest vectors for NruI sites (i did not get the full vector sequence from the lab were it was generated). Anyway i will try both suggestions as they sound both good. thank you!