Trouble with real-time PCR standard curve - (Jul/19/2006 )
I am new to qPCR and have been using power SYBR green on the ABI 7500 sequence detection system and am running my samples using 9600 emulation.
I have managed to get a nice looking standard curve with an efficiency of 94% once but since then it has all been down hill.
When I run the standards (1x, 10x, 100x, 1000x and 10,000x) the neat sample doesn't come up. Neat is at a concentration of 1ug/L, and there is defiantly something there as I have nanodroped it and my dilutions are all working and coming up at the appropriate C(t).
My standards are in homopolymer boil proof micrtubes that are apparently not sticky to cDNA. What is even more bizarre is that if I exclude the neat sample and put the 10x as the first value then this one doesn't come up.
I have repeated everything making new dilutions and being extra careful but the problem is still persisting.
I was previously using Roche's lightcycler and never had a problem with the standards or my primers.
What am I doing wrong???
dumb question...is it always in the first wells of the machine? if it's your first sample...could be mechanical
Its not always in the first well, In fact I have been putting the standards all around the 94 well plate as that was my first thought!
I'm sorry, that was the only thing I could think of...the way it's always the first sample simply seems like some sort of key to the whole mess..what if you run the curve in the other direction?
HEY wait a minute, are you telling the machine/ABI software to calculate the standard curves for you? what happens if you just put it in there like any old regular sample plate with all the default settings, then you take the Ct's and make the standard curve yourself? will it pick up the samples then?
I had a similar problem with my real-time RT-PCR. Something in the cDNA reaction inhibited PCR in the higher concentrations of the dilution series. Precipitating the cDNA reactions and resuspending them in distilled water before PCR eliminated the problem for me. Hope this helps