Hi Jenna,

Your LR reaction is not working and you are only observing the "background". There are almost always some colonies with empty plasmids, but it usually does not matter. Surviving cells with the pDest8 might got a mutated version of the plasmid carrying an inactive ccdB gene. Have you checked your entry clone by sequencing? Have you checked the concentration of both plasmids and their quality on an agarose gel? How long did you incubate the reaction? There are different improvement possibilities. You could start by using another destination vector to ensure the functionality of your LR reaction mix and the entry clone. When we faced problems with the gateway system, they were often caused by the plasmids. Increasing the incubation time of the LR reaction is another possible improvement, but will only lead to a slight  increase in efficiency.

Good luck!

Hi Boas,

Both vectors were at the recommended concentration and also showed relatively sharp bands on a gel. I haven't checked the entry clone by sequencing (I actually just sent it in today), but I have checked it by PCR and my gene appears to be in the entry clone. I only incubated for an hour, but that has seemed to work in the past. I've also transformed just the pDest8 into cells, and none of them survived. So at least in the negative control ccdb appears to be active. Do you have any other suggestions for improvements or optimization? Thank you!

Hi Jenna,

I would recommend waiting for the sequencing results. A mutation in one of the recombination sites could inhibit the LR reaction or at least reduce the efficiency.

Hi Boas,

I want to asked one question, after LR reaction, is gene orientation always correct ? Which is the best methods for checking the gene orientation after LR, because after LR reaction, I did both colony PCR and Plasmid PCR, but in both I saw many bands in gel and my cloned gene band intensity was very less. Boas Pucker

Hi Sandeep, I never faced any issues with wrong insert orientation after LR reaction. Due to the two flanking sequences, the insert should always be in the right orientation. Sequencing the plasmid is the best way to validate it. A colony PCR will only give you some hints about it. Multiple bands in a colony PCR indicate unspecific conditions. You could try to increase the annealing temperature if possible. Cheers, Boas

Thanks Boas, for your valuable suggestion