DNA purification from blood
Posted 21 October 2010 - 02:54 AM
The first thing I want to ask for is that one of my colleagues has used the salt method to purify DNA from blood, and the resulted DNA was not in good concentration and contained some brownish color (blood contamination). I was thinking to use phenol to repurify them. Anybody knows any way to repurify These samples since they already have a part of the DNA already extracted.
Secondly, i will really appretiate it if anybody can provide me with the standard protocol for purifying DNA from blood using phenol.
Thank you in advance
Posted 21 October 2010 - 05:21 AM
Purification by Phenol-Chloroform-extraction: nucleicacidsolution is extracted with one volume phenol (pH 8), After that with One Volume of phenol-Chloroform-isoamylethanol (25:24:1 v/v), After that with One volume chloroform. In between centrifugate and take topmost aqueous phase (without interphase) to new vessel. Proteins are denaturated and accumulate in organic phase, DNA stays in aqueous phase. Final Preciptation with ethanol will remove phenol. Don't use polystyrol or polycarbonat made vessels when centrifuge. They cant stay organic solvents. Phenol is toxic! Beware of Vapours.
Edited by ksturm, 21 October 2010 - 07:34 AM.
Posted 21 October 2010 - 06:41 AM
Thank you so much for your reply!
regarding the brown color in the blood sample; If i understood what you mean is that just centrifuging the tube to get rid of the blood right?
Do not you think that if i centrifuge the tubes i will get the blood and the DNA in the bottom so it will be hard to seperate them isnt it?
Posted 21 October 2010 - 07:29 AM
Next time doing a DNA purification from blood, think about eliminating erythrocytes. They barely contain DNA, so PBMC may sufficient for your expermient. You can get them by density gradient centrifugation with ficoll. actually there are very good kits for DNA preparation from blood (pharmacia, qiagen, promega).
Edited by ksturm, 21 October 2010 - 07:32 AM.
Posted 21 October 2010 - 08:57 AM
Edited by Maddie, 21 October 2010 - 08:57 AM.
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