BAC DNA purification - (Mar/01/2018 )

Hi all,

I am trying to purify BAC DNA (106 kb) using Qiagen large construct kit. I am able to purify 5-10 μg DNA (measured by nanodrop) but when I run it on agarose gel I see a band near the well. When I contacted Qiagen they told it might be genomic DNA contamination. I have attached the picture of the gel can anyone suggest me a solution?

Thanks,

Kirupha

So what are we looking at here - 

What % gel? How big is your BAC? What is the ladder (or size of the highest MW band)?

Hi Bob,

Thanks for the reply. It is 0.8 % agarose and the ladder is O'GeneRuler with highest size of 10 kb. The size of the BAC is 106 kb (which I have mentioned in previous post) and I loaded 2 μg of different preps. I would like to use it for microinjection into mouse embryo.

Thanks,

kirupa

Sorry I missed the size initially.  

0.8% probably isn't low enough % to adequately resolve a band of 106 kb, but your prep seems to have a reasonably strong band that could conceivably be at about 100 kb. Ideally you would use a ladder that is has a size range comparable with your expected band.  The bright smear up the top is indeed probably genomic DNA contamination in this case. 

I would attempt a gel purification of the band you see above the ladder.

Thank you very much for your suggestions bob. I will try to run a low % gel and also gel purify the band and see.

Thanks,

kirupa