real- time PCR is going to make me crazy! I just started with the method in january.
Here is what I do: Real-time PCR wirh Taqman probes and primer for 16S rRNA. They have allready been used succesfully.
What I do: I run my samples in triplicate. So on a plate I load my my PCRmix (with probe and primers, everythings) on the fist column (72 µL) and then add the samples (3µL). Then I mix with pipetting and divide it in the next 2 columns. So I should have (25µL) for each triplicate.
I use filtertips, multichannel pipet, work on ice and centrifuge my plate to avoid air bubbles.
I use different dilution of the same sample. And do each time a dilution series from a standard stock of E. coli 16S rRNA.
I have différents kind of problems:
First I do not have the same Ct number between my triplicate. (I have check my pipetting and it seems to be ok)
Second I have worst results now after a few try!! Now out of the triplicate I have very often only one wells with amplification! And nothing in the other. I do not understand how it is possible since I should have the same in the three triplicates.
Last, since I am starting the methods I always use the same samples. And the results make no sens: Between dilution I do not have corresponding difference between my Ct number. And between run. I also do not have the same number of copies at all.
So if anyone has an idea of what I do wrong, or what I should do... or Should not do. Thanks a lot. If you need more informations, please ask