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DNA extraction from cerebrospinal fluid using trizol

dna extraction trizol method low yield dna precipitation

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#1 Yasar

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Posted 26 December 2011 - 07:28 PM

Hi,
A happy Christmas and new year!
I am using TRIZOL to extract DNA from human cerebrospinal fuid (CSF). Generally, the number of cells in the CSF are very low (100-1000 cells/ml) and I am having trouble with the yield and quality of DNA. I use 800ul Trizol, 1 ul glycogen just before ethanol precipitation, and Na-acetate to wash the pallet. Then I use 75% ethanol to remove the salt and then resuspend in water. In the original protocol, resuspension should be with 8mM NaOH. Is this necessary or can I use water to resuspend?
Today, I made a mistake and instead of using 75% EtOH, I used 25% EtOH to wash the pellet. Since, the quantity of DNA is so low that I usually cannot see a pellet, and neither I can use a spectrophotometer to quantify the DNA, I am not sure if I have lost the DNA.
Besides this, does someone have a general advice to increase the yield of DNA from extremely low number of cells? These are human samples and I cannot play around.
Thanks
Yasar.
P.S. In Na-acetate wash step, can I just do it once instead of two washes? I fear with each wash step I will loose DNA. Also, would 10% ethanol in Na-acetate solution not dissolve the DNA?

Edited by Yasar, 26 December 2011 - 07:51 PM.


#2 akhshik

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Posted 28 December 2011 - 11:05 PM

hi there,

first of all you have lost most of your DNA by using 25% EtOH.

what do you wanna do with your DNA after Extraction will tell that which protocol you could use. is it a normal PCR or you wanna do some SNP with that?

and the last advice: search for forensic DNA extraction protocols, they are wonderful specially when you have limited source of cells.

#3 Yasar

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Posted 29 December 2011 - 07:52 AM

Hi,
1. I have to do a Taqman PCR on these samples, but will need to do SNP on some special samples after the PCR. Is a different type of DNA needed for SNP array?
2. I have cerebrospinal fluid samples banked in Trizol. I have about 600 samples already collected. For new samples, I have already started using Qiagen DNA Micro kit which is optimized for small quantity of samples.
3. I want to optimize the method for extraction of the samples already stored in TRIZOL. I have a very low yield of DNA and a very low 260-280 and 260-230 ratios.
Cheers.
Yasar





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