Hello everyone!
My problem is that unfortunately I am getting a good Ct value in NTC (NFW). I have tried three different primer sets (Target of three different genes) with NFW from different sources and surprisingly each primer set returned with a good Ct values.
Believing that SYBR-green (fast) could have been contaminated so, I used a totally new SYBR-green reagent yet there was no significant change observed upon RT-PCR.
I ordered new set of primers for same genes thinking that might be stock primers were contaminated and repeated RT-PCR with new SYBR green and fresh NFW yet I see there is very mild yet not a big difference in Ct values.
I use gloves and filter tips all the time.
I will be very grateful if anyone can give me some useful suggestion or insight to fix this never ending problem.
Reaction volume per well= 20ul
Primer conc.- 1uM each (FP/RP)
amplicon size ≈ 70-80 bp
Gene 1 Ct= 19
Gene 2 Ct ≈ 26-28
Thanks!
Kahin na kahin toh contamination hai kisi reagent mein water ya Primers mein ya sybr mein ya rod mein, if used. Bas! Nothing else. Also if NTC Ct is beyond 45, almost equals none.... Risky but okay. Sterilize the machine, bench and pipettes. Water not expired! Stupid but worked for me somehow 😇
Most common source of contamination is your micropipettes. Clean your work area, micropipettes, and throw away ALL of your reagents (buffer, water, primers, etc.). Make everything new (as if you were setting up a lab for the first time, don't use anything already made).
Also, don't use the same micropipettes to set up PCR that you use to handle amplified DNA. You can dilute a PCR reaction 1:1 billion and still get amplification.
Good luck!
Well qPCR NTC contamination is a very common problem as the qPCR is a very sensitive technique. I will recommended you dedicate one set of pipette for DNA and make the NTC first without open any DNA in the vicinity. It helped a lot for qPCR. Also autoclave the PCR tube. Aerosol contamination is like a ghost.
If it No template and not Negative Controls .
U may have to run a revalidation and re calibration for your equipment.
Majority of contamination occurs from the surfaces that we work on. Make sure you are sterilizing everything with 70% IP and also autoclaving everything. Rest one thing you can assure is that the instrument is in right condition. Ask your fellows whether they are facing the same issue. Get the instrument serviced, as you are already using fresh sets of everything. All the best.
You have already tried changing SYBR-green and primers, contamination might be due to water used in the experiment.
Do the melt curve analysis of the results. the curve in the NTC and the product don't match means, I think you might consider the results without worrying. once clarify this with some colleagues as well before considering the results.
It seems your work station and entire room should be decontaminated once fully. Also, if possible do clean your air conditioning instruments too.
Thanks to everyone for your valuable suggestions. I followed your suggestions but the issue was with high primer concentration. As I understood that my gene of interest expresses very low in mRNA pool and the primer concentration I was using was way too high (1 uM), I experimented by using different concentrations of primer (ranging from 0.2 uM to 1 uM) and found that 0.25 uM (micromolar) concentration of primers works best in my case.
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