some times my standard DNA also gives me weird curves..... I use taqman probes for standard curve analysis... following are some pics attached showing result of qPCR run. Can anybody help me with this??
Dear Milind,
Your Taqman qPCR amp plot appear to indicate that somehow the amplification reaction has not taken place at all. The deltaRN values are too low to be considered as true signal. This may be attributable either to faulty qPCR base reagents, improperly designed primers/probes, improper reporter/quencher dye combination, faulty thermal block or error in thermal cycle program profile.
First be sure that your primers are working. this you can check by running a conventional PCR with your qPCR primers using a well characterized DNA template as positive control, and check the presence of amplicons in agarose gel. if you have, you can also electrophorese the products from the above qPCR run, from which you have uploaded the figures.
After ruling out the all the faulty reagent factors, check the intsrument and thermal program.
Good luck
wishes
SD
The information you have given is not enough. Did you check if you have gotten any product by running the amplified reaction on the agarose gel?
In the cycling protocol, hot start at 95deg for 10 min to activate the DNA ampliTaq Gold is very important. If you miss this step.. you will not get any amplification and get wierd curves (like the NTCs).
Secondly, if your 2X PCR master mix is freeze thawed multiple times, or left at 4deg for more than 3/4 weeks, then you will see this happening.. Whenever you get a stock bottle, make sue that you make aliqouts in 1.5mL eppendorf tubes and thaw them as needed.
Thirdly, Always keep your cDNA frozen, and thaw it as an when needed. if you have stored cDNA at 4deg. for an extended time, it is possible that the DNA has gone 'bad'/degraded and you will get such weird results.
I hope that you have also validated the efficiency of your primers.
Hope this will help you resolve the issues. Good Luck
Looks like it is just low-signal background noise. It does not look like your positive control is working. I see samples that bounce around below the threshold of detection (the Ct line) or now called Cq line. It means that they are not amplifying. Check to make sure you can get your positive control to amplify using conventional PCR or with qPCR. If you can't get the positive control to work then you have a problem with either your positive control or one of your reagents.
Dear Carly,
My positive DNA works but since last two runs is not getting correctly amplified. Today I ran standards only and I got following result... curves are not sharp but there is amplification...
There could be an issue with your data collection point. Ensure the data is being collected following each cycle.
Hi, obviously you have a problem with your qPCR reagents, settings and/or thermocycler itself.
to test if the problem is the machine or your reagents I would try this reaction (and if possible another qPCR reaction you did in the past and worked successfully) in a different machine. If it works, machine was the problem. If it does not, reagents or settings.
Hope this helps,
Good luck!
Alfonso
I agree with Alfonso. As an additional suggestion, in case this is a RT-qPCR experiment, always check the quality of your RNA after extraction. Avoid freeze and thawing RNA: make aliquots and store them at -80º. I hope this helps!
Ruth
How much of template are you adding in each well? Less DNA might be one of the reason but most likely you have to quality check all the reagents as suggested by others.
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