ccdB Survival competent cells

[yanjia...@gmail.com]

Hi,

I have used Stbl3 for final ligation transformation, and got no colony at all. I even include a positive control that transformed with original TALE backbone vector, no colony either. I then double check the plasmid information from Addgene, and realize all the TALE backbone vectors contain ccdB gene, which is suppose to be grown in ccdB resistant strain. I am kind of confused since it seems like all the other groups just use the regular competent cells (Stbl3, Top10, DH5alpha) and get it work. Do I need a ccdB survival competent cell for the transformation? If so, which one do you recommend? Thank you.

Best,

Yan

[Feng Zhang]

Hi Yan,

The backbone vectors need to be propagated in ccdB survival cells. Your ligation should be transformed into conventional competent cells such as Stbl3. You might try using transforming DNA from the ligation reaction and also plating more cells. It is possible that your ligation efficiency is very low.

Also, you can run your ligation reaction out on gel and make sure that DNA fragments are being ligated together.

Feng

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Feng Zhang
Core Member, Broad Institute of MIT and Harvard
Investigator, McGovern Institute for Brain Research
W. M. Keck Career Development Professor in Biomedical Engineering, MIT
zha...@mit.edu

[Yan Jiang]

Thank you, Feng. I figured it out, I used the wrong plates with both Amp and Cl drug selections. I think it is the Cl killed all the cells. I will try the transformation again on Amp alone plates, hope it will work.   Yan

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