PCR master Mix problem...Plz help me out - PCR (Oct/30/2005 )
Hi,
I have been strucked up due to no amplification of the PCR primers which even worked earlier.
Please help me out.
When I prepare mastermix, I am not getting any amplifications but when I add the individual components to each tube seperately, it is working. I am not able to understand this, beacuse I was making mastermix earlier and distibute it to all the tubes. It was working fine. But all of a sudden it stopped working for me. I am not able to think of what the problem is.
I am doing this mix preparation as follows under sterile conditions.
10 X PCR buffer - 5 micro L
15mM MgCl2 - 5 micro L
dNTPs - 1 micro L
primers - 1 + 1
SD H20 - 36.5
Taq - 0.5 micro L
I will add all these components in the same order as I mentioned now in a sterile tube.
Please help me out so that i can have some progress. I changed all buffers and dNTP mixtures.
That is very strange. About the only thing I can think of (aside from the obvious -- no templates in the individual tubes, making a scale-up math or pipetting error, forgetting a component, etc.) is that perhaps the Taq sticks to whatever the tube you're using for your master mix is made from?
yeah, it's gotta be the tubes; that makes the most sense
perhaps there's a new lot of eppy tubes and there's something inhibitory?
I suppose another possibility is that something has a wacky pH that is inhibitory? but if you have changed all components that's not very likely
your enzyme isn't old enough to croak or anything, right?
There should be no difference, you must be leaving something out of the mix or treating it differently. People make mastermix similar to what you describe all the time without a problem.
Are you vortexing the mm, then spinning down, before you aliquot?
Are you using thin-walled pcr tubes in your thermocycler?
The mm method should have less pipetting error, not more.
-Matt
i have the same problem and i don't know what to do. can you tell me what did you d o to fix it?
I think you're making a basic error somewhere. There's no reason why that shouldn't work.
forgive me if this sounds lame, but the enzyme is typically very heavy and usually sinks to the bottom of the master mix. Maybe it's just that when you're distributing the master mix among your reactions, you're not picking up enough enzyme with it.
do you add the components in the same order to the single tubes (ie when you don't do a mm)??
I always start with water, perhaps the high buffer conc is having an effect on your primers or dNTPs (though I don't actually know if this happens or not).
To test if it is something to do with the tubes, you could make your individual reactions in the same tube as those you use for your mm, and then transfer to your pcr tubes. If the pcr still works then you know it is something to do with preparing the mm and not the tubes themselves.
Another question- do you set up on ice?