Hey guys,
I was doing BAC Clone extraction for Chromosome Conformation Capture(3C).
We used alkaline lysis method to lysis the bacterium.
Then centrifuged and applied supernatant to DNA extraction procedure, in which we extracted DNA through Phenol-Choloform.
But the Gel electrophoresis showed the DNA we extracted was so small.
The BAC we aimed to obtain should be more than 150kb.
Has anybody else met this kind of problem?
And I digested it with RNase A. And the shining band was gone.
My puzzle is I have digested it with RNase A before Phenol-Choloform, why there is still much RNA?
Hi,
In fact, it is really dificult to see the "BAC band" in a normal gel because it is really big and normally it stays on the well or close to. I saw your gel and at least in the 3th you have BAC. I usually do a nanodrop quantification and also a PCR specific of the BAC, a resistance or something like this.
During the extraction I recommend you to cut the end of the tips because BACs are not really stable and the friction of the tips is not good.
Good luck!
try using Machery-Nager Nucleo Bond BAC 100 plasmid purification kit (maxi AX 500/ BAC 100).
Henar is right, it's hard to see such a big band. Also, it's hard to nanodrop because such huge fragments of DNA take ages to resuspend and you might have to dilute a lot to be able to quantify accurately (especially if you want to made an equimolar mix for 3C)
thank you a lot.
I think you are right. But in terms of quantification, I was planned to do real-time PCR.
What do you think about this idea?
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