PCR = products come randomly? - (Oct/20/2008 )
Hi!
Maybe someone can find a explaination:
I did have too much noise in my qPCR, so I diluted the cDNA four times and run a real-time PCR. I had less noise -fine!
BUT, in each of my three replicates I got a different product (Tm: 80,0°; 82,6°; 84,35°C). I repeated it with another sample and again I got three different products (80,5°; 83,25°; 84,9°). This time also the NTC raise up but after 32 cycles and with lower melting temperature (Tm: 77,5°C).
Can anyone explain it to me? It can not be a contamination because I made a mastermix for all samples plus NTC and just split them up before I added the template. How can one mastermix, one primer system and one template produce three different products?
The replicates behaved not niced at all and had very different Cts, which could be explained by the diluted cDNA and the thereby increased pipetting failure. Allthough I still had a Ct between 15 and 20 cycles, which is not very late.
Anyway this drives me crazy...
Maybe some can help.
Thanx
Jan
Are the samples going into different positions in the instrument? If not, there might be a problem with temperature control. If they're going into different positions, you've got a very strange problem... 
Are the samples going into different positions in the instrument? If not, there might be a problem with temperature control. If they're going into different positions, you've got a very strange problem...
Maybe someone can find a explaination:
I did have too much noise in my qPCR, so I diluted the cDNA four times and run a real-time PCR. I had less noise -fine!
BUT, in each of my three replicates I got a different product (Tm: 80,0°; 82,6°; 84,35°C). I repeated it with another sample and again I got three different products (80,5°; 83,25°; 84,9°). This time also the NTC raise up but after 32 cycles and with lower melting temperature (Tm: 77,5°C).
Can anyone explain it to me? It can not be a contamination because I made a mastermix for all samples plus NTC and just split them up before I added the template. How can one mastermix, one primer system and one template produce three different products?
The replicates behaved not niced at all and had very different Cts, which could be explained by the diluted cDNA and the thereby increased pipetting failure. Allthough I still had a Ct between 15 and 20 cycles, which is not very late.
Anyway this drives me crazy...
Maybe some can help.
Thanx
Jan
Have you run the products on a gel to double check whether they are different sizes?
Hi!
Thanx for your answers.
1. No, I work with a Corbett Rotor Gene 3000, a rotor based system, so it could not be the position in the instrument.
2. No, haven't yet checked them on a gel. You think of not finished products during the elongation? But then I would expect to find more peaks in the melting curve, wouldn't I? But checking them on a gel is a good idea. I'll put them on an agarose an SSCP gel to check for lenght and different products.
Anyway it get's more freaky. It was only half the truth I told. I'm just searching for a reference gene and this gene I mentioned before is one of my candidate genes. in these two experiments I also amplified seven more candidate genes and more than one produced several products.
The only thing I changed to my experiments before was to dilute my cDNA, so I made four times dilution series of cDNA, ran it in triplicates to see if there is an effect of the dilution and -TATAAAA!- everything was fine again... The real-time world worked again. Single peaks, late NTC and some primerdimers only in the high dilution steps.
But I worked with the same water, same buffer, same SYBR, same primers.
I do not have a clou what was going on and why it works again now. But this concerns me even more.
Maybe the PCR explaination of a friend said it best "products come randomly"
All the best
Jan