Hi all
I use a sybr green based assay .
i sawproduct at ct 34 in NTC any recommendation ??
Hi Fatma
- bands with low melting temp in NTC tend to be primer dimer
- Thst is a primer self annealing problem
- The best way to solve this problem is alternative primers which have been screened for primer dimer formation
- Sonething like IDT oligo design ok will help you
- https://www.idtdna.com/calc/analyzer
If you run real time PCR with SYBR green you might want to consider doing a melting curve analysis after your PCR run. Check the melting peaks you have to confirm if you have unspecific amplification, or contamination.
Hi Fatima
Firstly, check melting curve analysis and melting peak to detect contamination, non specific DNA or primer dimer.
Hi Fatima !
To the best of my knowledge, for SYBR Green Real Time assays, there should not have any product for the NTC samples (i.e. neither Melt Temp nor Melt Peak) because, which means no band for NTCs even when you run an electrophoresis gel.
This simply shows that there was contamination in the NTC samples. So, check for that.
However, when you also mentioned that Cq = 34 for the NTC, ----> primers are non-specific (for specific primers, Cq value should be less than 30). In this case, There was primer dimers in your NTC samples.
If the Cq value is > 30 and if the difference of the Melt Temp among a triplicate of your unknown samples is > 1, ----> non-specific primers, regardless of the Melt Peak . It is then advised to select validated specific primers from the literature to assay your targets.
Good Luck !
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Dear Fatma, I would like to address your query in a bit other way. You have now many suggestions, ideas as stated above, but really does it matter so seriously or you are on the total safe side in your particular case of delayed ct in NTC and that has to be carefully taken in account before thinking of contamination or primer dimers issues and move blindly to discard everything or resources you have in your hands and restart from fresh. It is not a case always as per my entire experience.
Generally, we can say that any ct value in NTC may associate with PD or contamination (your pipettes, your water, dust and bacteria), and theoretically NTC should give no ct, which I agree. Usually, if the NTC gives signal along with a proper band on gel, it is a problem and you need to locate the source(s) I just mentioned above. Otherwise, you will see it on and on. If your target (GOI) wells also share the same Tm products as appeared in your NTC besides your GOI peaks then it may pose problem, as your reactions are not much efficient because of this contaminant or whatsoever and hence here the GOI ct is also not accurate. But, what if your NTC gives ct and your GOI wells only show your desired products, so it is significant now and this is the point you should understand. Your technique to perform qPCR, or hands on will also count here. You need to be sure that your technique of handling is ok, you are not introducing undesirables into your system.
So, as a rule also, if difference between GOI and NTC ct is above 7 cycles at least so ignore it, as it won't affect anything. For example; if your GOI ct is 26 and NTC gives 33 so it is ignorable. It has no importance.
And also, in NTC any ct value less than 31, you must investigate this thoroughly. I hope you understood the philosophy by now.
Best Regards....
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