BAC Clone Extraction. - (Jan/28/2007 )

Dear guys,

I have made a BAC library, but have a lot of problem to extract DNA from clones. Does anyone have a good protocol for extraction of BAC plasmids with 100 Kb inserts? Thank you very much indeed for your help.

Best wishes!

Shaelynn

I've had trouble in the past with BAC extractions, tending to get a lot of impurities and really low yields with the standard alkaline lysis methods. I've started using a kit from Sigma (NA0100) which seems to be cleaner and more efficient..

Hi both, I find that the best Kit on the market is the Nucleobond BAC 100 Kit (740 579) from Macherey-Nagel. I tend to get better yield and purity using this. I have used the Sigma one you mention but found that the DNA didnt keep very well.

If you just want to do a miniprep screen then Copeland and Jenkins method works well.

See Liu P, Jenkins NA and Copeland NG Genome Research 2003 13:476-484

for the improverished lab

"more, longer and cooler" - is my lab's standard answer.

We would grow the cells at 30 Celsius for a rather long time. As long as 36hr in some cases, though the normal incubation time is around 24hrs. And we will grow up to 12L of cells if need so.

The standard alkaline lysis method is augmented by inclusion of lysozyme treatment, LiCl RNA percipitation, RNAse treatment. And occasionally pronase.