Hmm, do both constructs transform well into E. coli? It could also be a lethality issue. Any chance there is a cryptic gene in the 2000 bp region?

Maybe retry the transformation into GV3101 along side a construct with the same selection marker that you know transforms well. That way you can tell for sure if it is a cell issue or a plasmid issue.

I did have a plant transformation construct that had lethality issues in Agro, it worked OK for E. coli (cells were a bit small, but they did grow), but for Agro there would be pretty much 0 colonies.

Hi Amy,

yes, the transformation in E. coli was

successful (both). In the 2000bp is a part of a gene (not the whole one) .

I cloned two different constructs: the 2000bp promotor region with the goi and the other one without the goi.

Oh no, four constructs! I have two different genes, too!

And only the 2000bp samples cause problems.

I am also beginning to fear that there are lethality problems...

Everything seems to be well done, but ... the huge 2kb-amplicon get your plasmid extremely big.

I recommend you to look for cells with a high transformation efficiency and to continue using the chemo-competent and heat shock transformation method.

This question has generated a small but interesting debate with my colleague Carlos M. Rivero-Núñez, whom I thank for his expertise. If you need something you can write him a PM, He's very helpful.

You're right, it's really extremely big and that's why it causes all these problems, I think. Because normally a transformation of C58C1 and GV3101 always works very well.

Do you have any idea which strain I could still use?

Thank you very much for your advice and best regards

Hi. I agree with Judith .