Swongel

Hi everyone,

In preparation for a methylation analysis using pyrosequencing, I have designed forward and reverse primers. My forward primer will be biotinylated. Now, I understand that I also have to design a sequencing primer. My question is, will it not be possible to use my reverse primer as a sequencing primer rather than design a new primer for sequencing?

Would be great to get your comments on this.

Many thanks,

Hi all,

I would appreciate your advice on the following if you could please:

I would like to compare DNA methylation levels of samples from saliva and blood that came from the same individuals. I will be using the EpiTect Bisulfite kit to convert these samples and subsequently perform a High Resolution Melt Analysis (HRM) using Rotor Gene.

The problem I have is the starting DNA concentration for the saliva DNA varies and is very very low (i.e. ranges from [2.82ng/ul] to [22.3ng/ul]); however, the blood DNA samples are all equilibrated at 100ng/ul.  Normally when I do this type of work all my samples are at the same concentration level i.e. [100ng/ul]. Do you suggest I run the experiment with varying concentration levels? A colleague suggested to select the minimum concentration I have as a threshold (in this case [2.82ng/ul]) and dilute all the remaining samples (even those currently at [100ng/ul] down to this concentration level in order to start with a uniform concentration level and continue with the bisulfite treatment and HRM analysis.This would mean having all samples at [2.82ng/ul] :-s!

I'm a bit conflicted as part of me thinks 1) yes, it's better to start with a uniform concentration level, specially if I want to compare the two groups (saliva vs blood) and the other part thinks 2) no, whatever level of DNA concentration I start with the amplification of the DNA depends on the amount of reagent I have in each tube. I assume once the product is finished the fluorescence level would reach its plateau.

Any advice?

Thank you in advance