An urgent question! plz help me
(by ivanoov from gmail.com)
Sun Oct 29 07:44:23 EST 2006
I think it would be no problem to isolate DNA from theese samples.
Howeverb because of degradation of erythrocytes in the samples a lot of
hemoglobine will be presented in the solution which certainly will
inhibit PCR. As you cannot remove it by salting out procedure I suggest
a min. of 1 extraction with saturated phenol (then chloroform) and use
of glycogen as a carrier in the precipitation (for degraded DNA). In my
experience this greatly removes hemoglobin and yields readily
amplifiable DNA. Alternatively you can use any DNA extraction kit
specifically designed for blood.
> Dear Yalda,
> Freezing and ice crystal formation probably will have damaged the WBCs.
> Assuming your samples were quite sterile when they have been harvested
> and frozen, you should give it a try (probably you have not much
> choice...). Even if the DNA has been partially degraded, you still
> should be able to amplify fragments of genomic DNA (I assume you'll do
> so kind of PCR afterwards - how do exactly check for different
> genotypes - by sequencing or by melting curve?). To estimate the amount
> of damage, you might perform some "positive" control experiments and
> expose a few frozen blood samples for different times (eg
> 0,1,2,3,4,5,10 days) to room temperature and assay the effect on DNA
> yield and on the results in your genotyping PCRs.
> I suggest to aliquot your samples and store them in different freezers.
> Extracted DNA may be stored in ethanol or even dry as backup as well.
> Good luck!
> yalda zare wrote:
> > Hi,
> > I want to extract DNA from sheep blood with salting out method.I am going to study polymorphism of major genes affecting ovulation rate in sheep.unfortunately because of an error in the electrisity of the frizer the frozen bloods melted and I think they have been in normal temprature for 4 days or more .Now I want to know can I extract good qualityand enough DNA from those bloods ?May I face any problems?
> > Thanks
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