real-time PCR after restriction enzyme digestion - (Jul/11/2011 )

Hi,

I'm Korean student in master course. I first write this forum..;so, please understand my grammer level..;;

I have an one question. I performed real-time PCR after enzyme digestion.

In my experiment, primer set produces about 160bp product including EcoRI recognition site in pGL2-basic vector.

So, I want to obtain minimal amplification peak using real-time PCR after EcoRI digestion.

However, amplification peak is detected at 10~100-fold below compared with undigested control.

I don't know what is problem.. please solve this problem..on the assumption that the DNA purity and enzyme are no ploblem

please....

The only real scenario is that your digestion did not go to completion so that you have some undigested product that can amplify in the PCR.

bob1 on Mon Jul 11 23:47:48 2011 said:

Thank you for your reply

How can I obtain the maximum efficiency? I performed many different condition including concentration of template, several volume of enzyme or total rxn, time course up to 120h, and treatment of exonuclease III, but cannot observe significant difference compared with undigested control. I only observed difference until 1000~2000-fold. Indeed, this value changes 6-7 Ct in real-time PCR analysis.

Is there anything about analysis of restriction enzyme efficiency using real-time PCR that I can refer?

Thanks, have a nice day

Seho on Tue Jul 12 01:15:39 2011 said:

Have you tested the efficiency of the RE by cutting a known sequence, such as a plasmid? Did you digest in the PCR mix or in the buffer supplied with the RE?

Sorry, no idea. Have a search on Pubmed...

bob1 on Tue Jul 12 09:53:05 2011 said:

Thank you,

I performed gel electrophoresis after digestion of plasmid that I used in my experiment. In this case, I detected linear band on the gel. And, I digest plasmid in the buffer supplied with the R/E. Rxn volume is 50ul or 100ul, and 2ul of digested plasmid is used for real-time PCR with SYBR reagent.

hmmmm....;; Could low DNA concentration ( about 1ng to 10pg ) be a problem? Indeed, I cannot perform high concentration of DNA due to the nature of my experiment...

Anyway, thank you for friendly reply. It was good experience for my first communication with foreigner. Again, Thank you.

Restriction enzymes rarely (ever?) cut to completion. It sounds from your qPCR data that you are achieving 99+% cutting efficiency, which is probably all you can expect. I don't think further optimization of your cutting conditions will improve this much. Your dilute DNA sample is an advantage, not a problem.

hmmm.. thank you for your reply.

Have a good day~!