DNA degradation in BAC preps - DNase inhibitor? (Apr/12/2007 )

Hi all,

I'm trying to prep BACs and to test the prep I use restriction digestion followed by pulse field gel electrophoresis and also genotyping by using PCR pimers spaced along the sequence of the BAC.

The PCR reactions of the preps are all returning products at the correct size when compared to a genomic DNA control (and also the negative controls are showing no bands so I haven't contaminated the reaction). However, the PFGE experiments just show a smeared band at lower molecular weights which I'm assuming means that either the BAC is sheared during the prep (unlikely given the lengths I go to and the numerous different protocols I've used) or that I have DNase contamination.


Has anyone else had these problems? Can anyone recommend a DNase inhibitor of something to add to prevent degradation?

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well there is good old EDTA. 0.5M EDTA work fairly well on the DNA plugs i've made.

I have heard of people using aurintricarboxylic acid as a nuclease inhibitor. Never used that myself though.Maybe that's work.

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Are you adding 100 uM thiourea to your running buffer? There are a set of articles describing DNA degradation from Tris buffer breakdown products and their effect on DNA. Most problems arise in E. faecalis DNA or Pseudomonas, but perhaps you are having similar problems.

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Thanks to both of you, I'll look into these!

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do you freeze thaw your BACs ?
i store mine in TE at 4° for weeks and they don't degrade. if i freeze them, they degrade partially....

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No, I keep them in the fridge. As soon as I finish the prep I put them in the fridge, then the next morning I digest for 4hrs at 37oC and run overnight for 18hours, chilled to 14oC. I tried supplementing the buffer with thiourea but it had no effect.

I just can't understand why all the PCR detection i do comes out with perfect results, yet the PFGE is terrible.

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You could proteinase-K treat your DNA samples to remove DNAse (although you should also just be able to heat kill it, unlike RNAse). Are you autoclaving your running buffer? I'd suggest also sterilizing your gel apparatus (dilute bleach & 70% ethanol, e.g.) to make sure nothing is growing in it. Are the PFGE markers running well? What size fragments are you expecting?

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There are a couple of different BACs with sizes ranging from 15kb to 125kb. I've cleaned the apparatus with 2% bleach. The markers run fine. I'm running the gel for only 4 hours today. It won't give me any kind of separation but at least it may show me whther the DNA is degrading on the longer runs.

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i was advised to check my bac clones by pcr supplementing 1.5mM Mgcl2. briefly, using a taq i amplified my clones and see if a product occured. After that, i confirmed by sequencing.
is this that aim you do with the gel ?

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The gel is just a rough 'genotyping' to confirm the presence of those sequences. The primers are pretty much randomly situated throughout the BAC and products compared to a Hela cell genomic DNA control. In the long term, i think we intend to use southern blots to properly charcterise the BAC, as we would like to use modified BACs to develop stable cell lines.

However;
I get DNA from the preps,
the PCR reactions match up perfectly with controls,
But I only get low molecular weight smears in my PFGE.

I actually ran the PFGE for 4 hours to see if it was the pfge process degrading the DNA and I still saw low molecualr weight smears! It must be in the prep process somewhere.

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