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BAC DNA purification


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#1 kirupha

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Posted 01 March 2018 - 12:56 PM

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

Attached Thumbnails

  • agarose gel.jpg


#2 bob1

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Posted 02 March 2018 - 07:02 AM

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)?



#3 kirupha

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Posted 02 March 2018 - 07:57 AM

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



#4 bob1

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Posted 02 March 2018 - 08:59 AM

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.



#5 kirupha

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Posted 02 March 2018 - 09:08 AM

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






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