Cloning with BAC - (Oct/28/2007 )

Hi,

I am trying to clone a tandem array which I am trying to generate in pUC18-backbone. However after certain repeats of array the bacteria (DH5alpha, JM109, SURE, DH10B...) did not survive, meaning it occurs recombination. I also tried to grow them at lower temperature. It helped for one round of cloning but at the current round of cloning, recombination happens.

So, I want to continue cloning in BACs as vector but I haven't work with them before.
I asked about this topic earlier and Perneseblue suggested I could use pBACe3.6. However, I am using BamHI, SalI sites (and XhoI should not be present in the vector) and this does not fit to pBACe3.6.

So, which BAC vector would be appropriate and where can I buy it? Is it expensive?

Thanks in advance

You may not really need a BAC. A low copy plasmid, with a p15A or pSC101 origin of replication would probably work almost as well. I believe Stratagene sells BAC plasmid kits. I think we have a set of plasmids that might work for you, including pBeloBAC-11 and supercos-1, but I'd have to check the freezer. We also have p15A and pSC101 origin plasmids.

Hi,

thank you very much. I would be very very happy .

I have another question. Do you think those vectors would work if I want to insert luciferase gene (with a regular promoter) downstream of the array and if I want to generate a stable cell line?

The array contains repeated sequences of DNA-binding site for thyroid hormone receptor and functions as enhancer element. So with the promoter and the luciferase gene downstream of the array, I can check the functionality of the array by luciferase assay. Then I would like to make a stable cell line for further experiments.