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Can I concentrate already extracted DNA with an extraction kit!?


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9 replies to this topic

#1 firefly2280

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Posted 21 February 2014 - 01:48 PM

Hey all

 

Odd question maybe but I have some extracted genomic DNA and need to pool and concentrate the samples. I have already submitted my PhD thesis so there isn't any money left to buy a gDNA clean up kit... So I was wondering if pooling them and then using the DNA extraction kit and columns I have then eluting in a smaller volume might work?

 

I'm only worried as you have to add lysis buffer to get the DNA to bind and wondered what that might do to the DNA. 

 

I checked and cant use PCR clean up kits etc (not that I have one) as they are optimised for smaller fragments. and I dont have one. Also, ethanol precipitation might work but Im not sure I have the reagents and the DNA in my samples is REALLY low. 

 

I would appreciate your thoughts please

 

Thanks



#2 hobglobin

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Posted 21 February 2014 - 01:56 PM

it's cheaper and less difficult to use a precipitation method with ethanol or isoprop such as described here: http://openwetware.o...A_Precipitation


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#3 firefly2280

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Posted 22 February 2014 - 04:57 AM

Thanks. I just don't have any sodium acetate. I can ask around but i'm the only one doing this kind of work, so Im not sure there will be any! 



#4 hobglobin

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Posted 22 February 2014 - 05:36 AM

Ammonium acetate as an alternative also works.


One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.

That is....if she posts at all.


#5 phage434

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Posted 22 February 2014 - 09:54 AM

Or plain salt.



#6 firefly2280

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Posted 22 February 2014 - 10:04 AM

Thanks! Thats a good tip. So 3M NaCl?



#7 phage434

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Posted 22 February 2014 - 10:17 AM

Yes, it is the sodium (or other positive monovalent) ion concentration that matters.



#8 perneseblue

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Posted 22 February 2014 - 05:25 PM

Or you can put on sample in a spinvac and simply evaporate all the water away.

 

Or put leave your sample on a table overnight and let it dehydrate (assuming the air in your lab is really dry and the samples size we are talking here is small).

 

Or a minute or two in an 50 degree incubator for a few minute and let the water evaporate. DNA should still be okay. After all protenase treatment does use 50C incubation to clean gDNA.

 

And if you do down the ethanol precipitation route, you can add some carrier to your sample to help precipitate the DNA (ie glycogen, dextran) You only need a very small amount.


May your PCR products be long, your protocols short and your boss on holiday

#9 firefly2280

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Posted 27 February 2014 - 05:20 AM

Thanks everyone for the tips. Will try the salt this weekend! 



#10 Trof

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Posted 27 February 2014 - 05:40 AM

I didn't wanted to wait for precipitation of 10x volume of elution buffer I mistakenly usead insted of 12 ul, so I put it in heatblock on 99 degs with opened lid covered with a wipe (we don't have a speedvac and larger volumes don't evaporate as well at lower temperatures). Didn't do a thing first 10-20 minutes, but then it just dissapeared after another 5 minutes like a ghost, while I was having tea. I was affraid it would be overdryed (and bit oversalted, since there was 10 mM Tris), but I sanger-sequenced that DNA just fine. Not exactly GLP, but obviously it can work too.


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