I've done a qPCR reaction using Bio-Rad CFX Duet, 10 ul reaction, using EvaGreen dye.
as you can see, image 01 is what I think as a good amplification plot.
Today I do the qPCR again using a home-made qPCR master mix (also using Taq Polymerase produced in our lab).
Here is the recipe for it:
20 mM Tris.Cl pH 8.0
10 mM (NH4)2SO4
10 mM KCl
2 mM MgSO4
0.1% Triton X-100
200 uM dNTPs
1X EvaGreen
300 ng of Taq Polymerase (in 10% Glycerol, 25 mM Tris.Cl pH 8.0, and 500 mM Imidazole)
5% Glycerol
a week ago I using the same recipe for the assay, but it show great amplification plot
but today, it show amplification plot like image 04 (sorry i haven't moved the data yet so i just using Paint to show what the amplification plot looks like)
what can cause the weird shape of it?
Hi Jonathan Jonathan ,
I'm pretty sure it is not this but I rather ask,
could it simply be that you forgot to log trransform your data ?
Another lead: try moving the baseline start of your amplification (most qPCR sofware have this feature). Sometimes it can greatly change the shape of the curves.
I'm curious, how do you produce your own Taq ??
Hope you'll find a solution.
Philippe.
Dear Philippe Paget-Bailly ,
Sorry for not updating the question, I've already found the cause of that weird plot.
The Imidazole concentration causes it I believe. the "weird" plot contains around 100 mM Imidazole, while I repeat the same assay but dilute my Taq at the point where the final imidazole concentration is about 20 mM everything works fine.
And for my taq production
I actually using a mutant of Taq polymerase D732N
which you can find in the article below. but I also modify it a bit by fusing it with other proteins for my research. I simplify the purification process by not doing dialysis or other chromatography method other than IMACArticle A Single Amino Acid Change to Taq DNA Polymerase Enables Fas...
Jonathan Jonathan god damn PCR inhibitors... :-D
Thanks for the Taq info !
- Badges
- Science method