Real Time gDNA allelic discrimination - help needed - (Dec/03/2007 )
I'm a newbie to this topic and I have a big problem regarding real time (allelic discrimination). My control DNA samples are wild type and mutated DNA fragments cloned into a plasmid. However, I am supposed to do the assay on genomic DNA. I would like to somehow overcome the difference in genomic and plasmid DNA amplification (plasmid DNA amplifies even at 50fg!). Can you tell me what would be the proper amounts? (e.g. 50 pg gDNA = 50 fg plasmid DNA or something like that)?
Also, what is a typical amount od gDNA used in allelic discrimination assay?
The second problem is that I cannot get any reasonable reproducibility in my duplicates. I tried fresh pipettes unused by anyone else, changing the volume from 1 ul to 5 ul – it didn’t work (I got a satisfactory result only once, and it was with 1 ul DNA, but then I had to make some changes in the protocol, since my mutant primer was hybridising unspecifically to WT DNA - ever since then I could never get such a result).
I think I've tried to get all of the variables stable... Please, help! :-)
I guarantee I will have more stupid questions in the future... :-D
Celera estimates 6.6pg gDNA per cell which is 3.3pg gDNA per gene copy (assuming only two copies) so for each ng of gDNA you have 303.3 copies of a normal gene.
50 fg of plasmid = some number of copies dependent upon plasmid size (sorry will have to look up the conversion factor tomorrow will get back to you)
Anyway if you already know your plasmid copy number then you have the information you need. High variability not due to pipetting error may be due to bad/inefficient primers. Check the efficiency of your reaction using a standard curve made of your plasmids, if you are worried about complex template (actually should be pretty similar anyway) spike gDNA with plasmid...
you make your duplicates up in one reaction and split them in half for duplicates right?
hope this helps and good luck!!!
Thank You for your help, I really appreciate it!
>you make your duplicates up in one reaction and split them in half for duplicates right?
No! Should I? I had no idea you are supposed to do it that way. I just add DNA seperately to each well.
Some time ago I've made a web form, that counts copy number from concentration, you can use it if you like. I used it for real-time as well.