I am currently using Gateway cloning for my project. I have DB3.1 which propagate the pDONR vector containing the ccdB gene fine. Just out of curiosity, as described on the BP clonase user manual, bacteria with F Episome are not suitable for propagating the recombinated product, as it contains ccdA antidote and disallow negative selection.
With this understanding, bugs with genotype F' should able to propagate ccdB containing vectors such as pDONR. However, no colonies form with JM109. Did I misinterpret something here?
Genotype of JM109 from OpenWetWare is
endA1 glnV44 thi-1 relA1 gyrA96 recA1 mcrB+ Δ(lac-proAB) e14- [F' traD36 proAB+ lacIq lacZΔM15] hsdR17(rK-mK+)
http://openwetware.org/wiki/E._coli_genotypes#JM109
yes JM109 should be able to host a ccdB plasmid. However be sure you have grown your JM109 stock on minimal so that you know it is still carrying the F'. You may have lost it otherwise which would explain your result.
As a slight aside gateway selection should not be transformed into NEB-turbo bacteria for the same reason. They are not however very good hosts for the destination vectors (I've those carrying ccdB). Stick with DB3.1s for this
Hi Nguyen,
pDONR vectors also contain CmR in the gateway cassette for chloramphenicol resistance. You may try to grow the obtained colonies on LB+chloramphenicol as the second confirmation. Chloramphenicol is heat stable and you add it to the medium before autoclave.
If you are not sure about your transformation, you should isolate the plasmid and confirm it by digestion. Enclosed you find the vector sequences for pDONR221 and pDONR/Zeocin. I do not have the sequence for pDONR201, but I think these vectors share the same backbone. Just try with EcoRI/EcoRV to see a fragment of ca. 700 bp on agarose gel.
Best wishes.
Tuan
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