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RNA carrier when using not so good DNA for bisulfite conversion


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3 replies to this topic

#1 lmg

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Posted 05 April 2011 - 01:30 AM

Hello,

IŽll be doing some bisulfite conversion and wondering if you recommend using RNA carrier (in EpiTect kit) when dealing with DNA of not so exceptional quality and maybe even with some pollutant (ethanol residua)?
DNA was extracted using NucPrep method 6 years ago, extracted from tumoral biopsies; having 260/280 ratio around 2,00, but very low 260/230. Unfortunately this is the DNA I have to use in order to perform MS-HRM for methylation analysis.

Thanks in advance,

lmg

#2 MoB

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Posted 06 April 2011 - 12:57 AM

Hello,

IŽll be doing some bisulfite conversion and wondering if you recommend using RNA carrier (in EpiTect kit) when dealing with DNA of not so exceptional quality and maybe even with some pollutant (ethanol residua)?
DNA was extracted using NucPrep method 6 years ago, extracted from tumoral biopsies; having 260/280 ratio around 2,00, but very low 260/230. Unfortunately this is the DNA I have to use in order to perform MS-HRM for methylation analysis.

Thanks in advance,

lmg


Ethanol will not affect the bisulfite treatment. Carrier is only recommended if the total DNA input amount is below 100ng and has nothing to do with the quality of your DNA. But it won't hurt... ;-)

Hope that helps...

#3 lmg

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Posted 14 April 2011 - 02:49 AM


Hello,

IŽll be doing some bisulfite conversion and wondering if you recommend using RNA carrier (in EpiTect kit) when dealing with DNA of not so exceptional quality and maybe even with some pollutant (ethanol residua)?
DNA was extracted using NucPrep method 6 years ago, extracted from tumoral biopsies; having 260/280 ratio around 2,00, but very low 260/230. Unfortunately this is the DNA I have to use in order to perform MS-HRM for methylation analysis.

Thanks in advance,

lmg


Ethanol will not affect the bisulfite treatment. Carrier is only recommended if the total DNA input amount is below 100ng and has nothing to do with the quality of your DNA. But it won't hurt... ;-)

Hope that helps...


MoB thanks. I used it. The next series i will try not to, just to compare. ;-)

#4 vilperte

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Posted 06 February 2013 - 07:04 AM

Hello!

I tried the bilsulfite conversion with and without carrier.
I started with 200ng and I ended up with 40ng (with carrier) or 3-5ng (without carrier).

So, in my case, even starting with more than 100ng, the carrier really helped to increase the yield!

But I'm still struggling with my PCR. I couldn't amplify any of my fragments (I have 5 different pairs of primers).


Good luck :)




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