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vilperte

Member Since 04 Feb 2013
Offline Last Active Mar 01 2013 05:17 AM

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Active Posts 19
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About me

Lab or Group
Molecular Biology
My research interests
Epigenetics

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Website URL http://www.genok.com

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Mimoza
01 Mar 2013 - 02:47
neuron
11 Feb 2013 - 21:33

Topics I've Started

Which DNA strand is recommended

11 February 2013 - 03:35 AM

Hello!

Since it is assumed that both DNA strands have the same methylation pattern, there is no need to sequence both of them.

But which strand is recommended to sequence? I've read that is easier to sequence the sense strand (using the reverse primer). It that correct?

Thank you Posted Image

Bisulfite sequencing PCR worked

08 February 2013 - 01:45 AM

Hello!

I've been reading a lot of topics here, trying to get as much information as I can.
After 1 month struggling with my BS PCR, I managed to amplify my fragments.

I've tried to convert my DNA again, I changed twice my polymerase, I tried gradient PCR, without any results.

What really helped was: decrease the extension temperature for 65C and increase the time for 2 minutes (even with fragments around 300bp). Increasing the cycle number can also help it.

So, thank you for all the people that has been postings tips and trying to help the others. It was really useful for me!

Now I can show some results for my supervisor!

Quality of Bisulfite Converted DNA

04 February 2013 - 08:02 AM

Hello!

I'm having some problems with my Bisulfite PCR. I cannot amplify the target fragment.
At first I thought it could the primers, but I went trough all of them to see if I had done something wrong. All the parameters look good!
I designed them using Meth primer software.

So, I'm thinking that it could be the quality of the converted-DNA. I converted it using the QIAGEN EpiTect kit.
I have quantified it using Nanodrop and agaroses gel. It looks fine, but on Nanodrop you can see a peak at 230nm (higher than on 260nm).

Does anyone know how to be sure of the DNA quality?

Thank you!