Hey all, I occasionally receive frozen blood in which I need to purify DNA from. If the blood has not been handled properly (properly means kept frozen to <-50°C in ACD) then on thaw the blood is no longer opaque. The level of white blood cells retrievable is small and there is considerable contamination with red blood cell pigments (based on appearance I assume that the WBC have been thorough opened up and these pigment containing compounds have been trapped by the white blood cells. After the K/Dodecyl sulfate precipitation small amounts of EDTA can be added to the clarified DNA containing solution, and this gets the iron/heme off the DNA prior to precipitation (altering the color of the precipitate from brownish to white), which appears to salvage the DNA for later PCR work. Although ominous some DNA can be purified from the blood, usually to the level of 10-20 ng per microliter in relatively small volumes, which is well below the amount I need for semi-quantitative based SSP-PCR. The use of EDTA has its draw backs, since it can greatly increase 260/280 and thus combinations of EDTA and protein can give the appearance of success, when little DNA has been purified. As a consequence I need to repricipitate the DNA and test to see if the OD 260/280 shifts and if it does repricipitate one further time to make sure the OD 260/280 is stable. Since genomic DNA is not particularly easy to get back in solution once precipitate at higher concentrations this is a very lengthy task.
I have been using GenomiPhi V2-DNA amplification kit to amplify these DNAs. What I have found is that if the DNA concentration is below 6ng per microliter I get no amplification. Between 6 ng and 10ng of DNA per microliter I get some amplification and best amplifications are seen at levels of 10 - 40 ng per microliter. DNA concentrations are based on OD 260/280 above 1.7 (usually 1.8 to 2.0). Above 30 ng there is no obvious increase in yield. This is the problem, the kit says that starting with 10ng of pure DNA on can obtain 4 to 7 ug of DNA. In my hands starting with 25ng the best I can obtain is around 0.6 ug of DNA the yield is about 1/10th of what the company states. Does anyone who uses genomiphi have the same problem? Are there any tricks to improve the purification. The latest lot I have has an assay date of October 2011 and the expiri date of February 2013 and I am wondering if the enzyme or the reaction buffer might have gone bad? Thanks Philip _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
