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DNA extraction...help needed! - extracting dna from buccal cells in ethanol (i think) (Jul/30/2008 )

so i just received some samples from the field from a non-geneticist and they were obtained by using cotton buds (q-tips) to swab the insides of the mouth and then dipped in a tube of ethanol and swirled around before being thrown away.

more than likely i will not be able to find dna in many of the samples. however, these are from a very remote area which i've been needing samples from for a while. so i would like to be able to get as much as possible.

does anyone have any suggestions or a link to a protocol for this type of extraction?? i'm typically used to extracting from saliva samples in lysis buffer or from blood or bone...have never tried this.

all the best....

-sparky-

Hi Sparky,
this sounds really challenging...
I can only recommend you to use a commercial spin-tube purification system.
Selecting the right product depends also on the size of your EtOH-sample.
As EtOH sometimes interferes with binding to the glass-fibre, I would first reduce the volume of your sample by evaporation e.g. in a speed-vac. Then add water (maybe with a carrier) (or the 1st buffer of your cleanup kit) again to resolve the dna which may have bound to the wall during EtOH evaporation... Maybe it may also help to put in some carrier-na, e.g. MS2-RNA (or another carrier which may not interfere with your analysis).
If you can reduce your sample volume to a small amount I can recommend you the new "micro" columns which are especially designed for small amounts of DNA and which allow you to elute with a tiny volume to get higher concentrated dna.
You can get them from www.roche-applied-science.com. They are called "High Pure PCR Cleanup Micro Kit". Also they are pcr cleanup kit I use them successfully for all kind of sample materials where you have only tiny dna amounts (which is also described in the pack insert).
For larger volumes you have to search for another suitable kit. The companies roche or qiagen have plenty for different applications...
Maybe you first create some additional samples by yourself with which you evaluate different kits and methods before starting with your precious samples!
Wish you good luck rolleyes.gif

-THE_PROFESSOR-

QUOTE (sparky @ Jul 30 2008, 10:41 PM)
so i just received some samples from the field from a non-geneticist and they were obtained by using cotton buds (q-tips) to swab the insides of the mouth and then dipped in a tube of ethanol and swirled around before being thrown away.

more than likely i will not be able to find dna in many of the samples. however, these are from a very remote area which i've been needing samples from for a while. so i would like to be able to get as much as possible.

does anyone have any suggestions or a link to a protocol for this type of extraction?? i'm typically used to extracting from saliva samples in lysis buffer or from blood or bone...have never tried this.

all the best....


Dear sparky,
may be this animation can help you
http://learn.genetics.utah.edu/units/biotech/extraction/

-fiat29-

Just centrifuge to get the most cells as possible, discard etoh and wash cells with PBS 1X then use the DNA extraction protocol for cell that you normally use. Is better to use PBS instead of water to avoid cellular lysis. Try that the person that takes the sample use PBS or another saline solution instead of etoh. The cells must be preserve undamage as possible and the etoh dihydrate the cells so maybe you are not getting enough cells because they are dying (they should be looking like raisins and maybe liberating proteins like dnase, so not much dna survive) and if possible just cut the cotton tip into the tube so you will have better chances to get the most cells as possible.

-merlav-

Thanks for the suggestions! (and the link to the animation...it's very kitsch and fun...and educational!)

Normally when I collect dna, I collect saliva samples in a lysis buffer using a protocol that a colleague of mine created. It works incredibly! But, like has happened before, these samples were collected as sort of an afterthought by a non-geneticist.

I think that I'll take the suggestions about spinning it down and getting rid of the ethanol first.

Two more questions:

They are in these weird shaped tubes which won't fit in a centrifuge...would I be able to transfer the contents (roughly 500ul) to proper tubes without losing any cells?
And if I then centrifuge them for a while at high speed, would I then be able to pipette out some of the ethanol so I can then let the remainder of the ethanol evaporate more quickly?
Someone suggested using the QIAamp DNA Micro kit since it' allows for elution with low volumes of buffer...

Thanks again!!!

-sparky-

I have not use the kit, but answering the question about tubes I'll sugest to transfer the cells to another tube (make sure the pellet is resuspend) and wash the tube with some buffer just to make sure you are taking all the cells possible. After centrifuge the cell you can decant or pipette very carefully the excess of ethanol and let the remain evaporate (if got a speed vac drier use it).

-merlav-