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No Band With Large DNA (BAC)


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#1 jms88

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Posted 26 November 2013 - 07:41 AM

Hey guys, I am new to this community, and was wondering if you could help me out with something

 

Recently received a BAC clone from CHORI and I am trying to "prove" that they sent us the appropriate BAC. Using solution based mini prep (not a kit) I have extracted the BAC, on spec it shows ~700ng/ul with a 260/280 of ~1.9. I have 2 BAC clones of different sizes, both are rather large (one is >50kb) - although I would need to pull up the exact numbers if that is important for you to help. Using GCK I have predicted the restriction digest for ECOR1 which shows a few small bp bands (one ~250 and one ~500bp). I have been running on a 1% agarose gel and my problem has been that I do not see any bands. The DNA ladders look as expected, but in the wells containing the digested BAC clone I don't see any bands. Looking back at my notes, I have been loading too little DNA for the 500bp bands to light up, but I can't figure out why the larger bands ~5kb (or larger) aren't showing up at all? I would expect there to be at least 20ng of the 5kb bands present in the well, and I am using ethidium bromide to stain.

 

I have also run an undigested sample which did not show any bands either.

 

I feel confident that there is DNA present in the well - what could be going on here?

 

Thank you for your time and consideration!



#2 AquaPlasmid

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Posted 06 December 2013 - 11:31 PM

700 ng/ul is a lot of DNA for a BAC miniprep! How much culture did you use for the miniprep? How much water did you use to solubilize the DNA? Do you mean that you saw DNA in the well but not in the gel lane for the uncut? Did you see any DNA in the well after EcoRI cut? If not, you might have gDNA contamination but didn't have the BAC.


Edited by AquaPlasmid, 06 December 2013 - 11:33 PM.





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