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An urgent question! plz help me

An urgent question! plz help me - Protocols and Methods Forum

An urgent question! plz help me - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 10-28-2006, 02:53 PM
yalda zare
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Default An urgent question! plz help me



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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  #2  
Old 10-28-2006, 07:26 PM
WS
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Default An urgent question! plz help me

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!

Wo

yalda zare wrote:

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  #3  
Old 10-29-2006, 12:44 PM
chovek69
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Default An urgent question! plz help me

Hi,

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.

Ivan


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