No replies to this topic

[Mimoza]

I need to create an unmethylated control using whole-genome amplification by φ29 which I have bought from New England Biolabs (NEB). My intention was to follow the procedure of Umetani et al, 2005, in order to create controls for my methylation experiments. Apparently, the φ29 polymerase is covered by a patent and the company wouldn't give me the exact protocol. I have been reading papers regarding φ29 polymerase, but there are some issues that I still don't have clear.

-People say that this method is very sensitive to contamination and that it is normal to have DNA in the negative control. This happens to me, but is this really normal??

-It seems that the DNA is amplified by measuring with the nanodrop, but after a standard ethanol purification step, all the measures drop to 0. So, I think that what the nanodrop is measuring at the first place should be more dNTPs than amplified DNA. What do you think? I am using a purification protocol which has been working in my hands for cleaning-up PCR products.

-The source of the DNA is a pool of fish larvae. Does this play a significant role for the amplification? Should I maybe start with something more "clean", like DNA from just a muscle?

-I have read that one should start with less than 10ng and elsewhere that one should use at least 10ng. Umetani (2005) is using 1ng in
order to have at the final product the least methylated DNA possible. With which quantity of DNA should I start?

Any help, advice, protocol....is more than welcome. I put Umetani's paper attached.

Thanks a lot guys!

Mimoza

Attached Files

•  Umetani et al 2005 Synthesis of universal unmethylated control DNA with φ29.pdf   623.81K   103 downloads