Hello all.
I have been working with a sequence that is a 571 bp clone, and I have a primer pair that gives a 150 bp amplicon. When I ran the system in real time for a calibration curve I got some results I could not explain. First of all, the highest concentration sample (1000 ng/ul) did not give a signal at all. The next dilution in the series (100) gave a signal of around 30, and the serial dilutions where around 1 or 2 increments (needless to say the eff. value is very high and basically unusable, around 300%). Raising primer conc., doubling SYBR green mix amount and lowering annealing temp had no real effect. Adding DMSO (5%) had a good effect on sample variability, but not on the Ct values. When I ran the reactions on a gel I got a very good product for the first 4 dilution, even for the sample that did not generate any Ct to speak of. Does anyone have an explanation for this lack of correlation between getting the product and not getting a Ct value? If you encountered something like this before, what steps did you take to improve the real time reaction?
Thank you
Ori Elad
HI
Do you have primer dimer? What is the melting temperature of your product?
best
An efficiency of 300% points to problems with your PCR when you use SYBR Green. If you hypthesise that all possible PCR products are syntesised from cycle 1 they all can duplicate a factor 2 maximally. As some products are formed later on for the first time, your efficiency will below 2! So a factor 3 is impossible.
If you produce non- specific products they will also be amplified maxiamally with a factor 2.
Next to primer dimers, you can easily get recombination of single stranded products after mispiming. These normally have a lrger size than dimers and even your desired amplicon. But these also amplyfy at a factor 2 t the maximum.
So an afficiency of 300% is really wrong.
Lowering annealing temperature, enhancing primer concentration etc all promote the synthesis of more non- specific products.
I am not sure if the bands you see in elektrophoreses are the desired amoplicons. How do you know you had "a very good product"? Did you sequence them? do they hybridise with a specifi probe? Otherwise a position on the gel might also indicate a non- specific product.
Did you do melting curve analysis?
Did you do gradient PCR?
But when you see no product at all; How was the setting of yourq PCR machine? Did you measure at the end of the elongation phase. Did you measure in the correct channel??
What is your input material? Is it a 571bp long DNA fragment?
If so, your 1000 ng/ul standard contains about 1.6 x 10^12 molecules per ul. Given that a single molecule would give a ct of around 34, one could expect the upper limit of quantification to be at ct 10 what corresponsd to about 2^(34-10) = 16.7 x 10^6 molecules. This is about 1-10 million times lower than the concentration you actually used.
So if I am correct you need a much, much, much higher dilution of your standard. The results you found are caused by the fact that the SYBR-Green signal is way above any reasonable threshold already from the first cycle(s) on. The software won't be able to recognise a typical exponential amplification curve (have you looked at the curves?).
The above suggestions are excellent - perhaps if you could show your gel, that would help. We did a brief comparison between CT and SYBR qPCR in Henderson, H. H. et al., J. Virol. 90 (2015):1231-1243.
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