Hi Hagen,

I came up with some idea about the PCR issue.

Since I have made many failures in the BAC transfer, I was increasing the amount of BAC DNA in the electroporation.

If I use 1ug of BAC for electroporation, even after the addition of SOC and plating, the concentration of BAC DNA seems to be high enough to be amplified (20pg).

Anyway I carefully extracted and purified BAC DNA from the growth of electroporated bacteria, and I finaly got a good PCR result.

Mini-lambda system seems not to need transfer BAC DNA. I don't know much about this sytem, but it may be a better way.

I appreciate your useful advice.

Kenzi

Hi Kenzi,

We have also tried a lot of recombineering of BACs using the SWXXX cells. In our experience the success was alway dependent on the BAC you used. If you are lucky and you have few repetitive sequences in your bac it works and if you are less lucky you are facing the problems you currently have. In most of the cases we found similar to Hagen that the BAC was fragmented thereby deleting most of the sequences required for e.g. your targeting construct. We have changed now our recombineering protocol to a system provided by gene bridges and this worked a bit better, although we still find from time to time BACs that do not behave properly.

My advice: Check the BAC with Southerns if you really have fragmented BACs or if your probs simply result from a not functioning PCR.

If you have fragmented BAC DNA change the strategy: use an alternative system (e.g. the mini lamda system or the one from gene bridges) or change the BAC.

Good luck xwith yuor experiments

Volker

Hi Volker,

Finally, I retrieved the desired construct.

I knew too well the importance of the BAC DNA quality.

Among a few different BAC miniprep methods I tried, which includes NCI's and modified QIAGEN midi kit, I found Valerius' BAC miniprep protocol is best.

http://valeriuslab.org/

It takes a bit of time to complete, but the yield and quality of BAC DNA are always good.

I might be lucky this time. We can get the imformation about a repetitive sequence from a genome database like EBI, but targeting strategy may leave me no choice to use different BAC.

If I face similar issue in next time, I may try alternative methods.

Thanks.