gateway cloning trouble: a lot of false positives colonies - (Apr/27/2006 )

help ..........

im using pdnor 221 vector , but all my recombination reactions lead to a big amount of false positives regarding in the fact that pdnor 221 have a ccdB lethal gene , is this an usual phenomena? , my pcr fragment is 1050 bp , is there any relation whith the fragment lenght and the low efficiency of my recombinations?

any advice in how to improve the gateway recombinations will be accepted ..........

strange indeed.... which kind of cells do you transform in? if they contain F' , then they are resistant to the effect of the ccdB genproduct, so plasmids without recombination will grow.

up to now, i hardly ever had any fals positives.

Hi there,

Plz, write to me about your recombination reactions parameters, and I will tell you what you must do!

M.Ragab
France

Hi everyone,
In our lab we discovered the same problem of false positive, not recombined gateway plasmids. Many of the pDONR vectors after BP reaction as well as the destination vectors after LR reaction still contain the ccdB gene (~ 50% and more). We use 2 ul of a BP or LR reaction for heat shock transformation of competent DH5alpha cells who should be sensitive against ccdB, but even if we transform the original pDONR vector we get at least 6 colonies (efficiency: 4*10^6 cfu/ug pUC19). In our lab protocol we decreased the amount of BP and LR clonase II to 0.8 ul per 10 ul reaction to reduce costs and usually still got enough clones with recombined vectors. Regarding to maximum efficiency we usually use 150 ng of linearised entry & destination vector and incubate at 28°C over night as stated in the manual. But lately we found more and more not recombined vectors, especially the ccdB selection after LR reaction fails. If anyone has an idea how to improve the efficiency of ccdB selection or how it is possible that the gateway system fails i would be very thankfull to hear it, if this system isn't reliable anymore it's not worth the time and money you spend on it.

Michael Gebert