Theoretical on Miniprep mechanics

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Andreas "Mega" Stuermer

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Dec 4, 2020, 7:18:42 AM12/4/20
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Hi everyone! 

I was wondering the following: 

During a miniprep, you first lyse the cells with sdoium hydroxide and then neutralize everything with aetic acid. 

Now a question came up that I never thought about: Will this not denature the DNA? During the high pH of NaOH there will be a lot of OH- groups floating around interacting with hydrogen bonds? 
Will the DNA not be partly or fully single-stranded then? 
 
Will it fully reaneal very fast, or not? will there be non-covalently joined and partly overlapping plasmid strands? 

Not that it really matters for routine applications  but I find it very interesting and would be interested if anyone has thought about this, knows literature about it or knows the right keywords. 

Thanks! 

Cathal Garvey

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Dec 4, 2020, 7:58:11 AM12/4/20
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You're correct: genomic DNA does get flash-denatured in a miniprep. That's a feature, not a bug! :)

A miniprep is intended to collect small, supercoiled DNA rings, which are able to resist this denaturation because of their supercoiling, so they can be collected in the supernatant fraction at one stage of the miniprep, leaving most genomic DNA behind.

If I recall correctly, the denatured genomic DNA is also effective at helping to capture some of the protein fraction, so it has the side effect of helping reduce that also.

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Dec 4, 2020, 12:18 by andreas.t...@gmail.com:
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Andreas Stuermer

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Dec 4, 2020, 8:24:06 AM12/4/20
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Oh interesting, thank you! I see, that makes sense! I'll need to read more on the mechanics

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Cathal Garvey

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Dec 4, 2020, 8:36:31 AM12/4/20
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It's a very neat procedure. Years ago I dug up some articles/blog posts about some of the basic "everyday" molecular bio procedures and minipreps were one of the ones that got explained really well - it was great because I hate doing something I don't understand, and I had been taught how to do minipreps "by following instructions", which isn't great.

I mean, you still got to follow those instructions, and you probably can't mix buffers from different kits (different buffer strengths etc.) but it's good to know what's actually going on, and it can help you debug things if the kits break.

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Dec 4, 2020, 13:23 by andreas.t...@gmail.com:
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