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High Concentration DNA Extraction


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4 replies to this topic

#1 marcus4545

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Posted 20 January 2011 - 07:45 AM

Hey, Ive been trying to extract a large amount of DNA while keeping the final concentration high as well, but this just tends to end up with very very sticky and even lumpy DNA, any suggestions on how to avoid this??
-The numbers im working with are something like 50,000,000 cells and ive been getting final concentrations of say 300ng/ul at most but i would like more idealy
-Been using a phenol/chloroform extraction

Thanks

#2 David C H

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Posted 21 January 2011 - 06:32 AM

The stickiness is probably from polysaccharide, due to having too many cells in your prep. Most protocols are calibrated to avoid this; adding more tissue or cells than recommended will give poor results. If you want large amounts of DNA, scale up your preps by adding more lysis buffer, etc. or doing multiple preps and pooling the DNA at the end (it is usually easier and faster to do multiple preps).

#3 David C H

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Posted 21 January 2011 - 06:39 AM

Forgot to add, if you want a high concentration, after pooling samples, precipitate with PEG 8000 and resuspend in a smaller volume. PEG purification can produce a very clean, clear pellet; be careful or you can lose it. You can precipitate with high salt (sodium acetate, etc.) but because the washes only remove a fraction of the salt, for very concentrated DNA you will also have high salt concentration.

#4 Maddie

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Posted 21 January 2011 - 06:39 AM

I don't work with cells, but I agree with David. With soft and hard tissues, increasing the amount won't help. It's better to either concentrate what you already have from a reasonable number of cells or extract multiple times, pool and concentrate. I think..
Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

A. Einstein

#5 pDNA

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Posted 21 January 2011 - 08:44 AM

i always use CTAB-based methods to extract the polysaccharides ...this helps to decrease viscosity!

Regards,
p




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