Dear all,
Actually, I am working with AriaMx Real-time PCR System. I read the protocol of this device and following that to work but unfortunately, I don't know why I can not get any results.
I mean that I mix the water (8 microliters), master mix (10 microliters), DNA template (2 microliters) and F & R primers (each one of them 2.5 microliters), and putting in the device. then, I set the regulation of the device ( as recommended in the protocol) and run the experiment. But after even 20 or 30 cycles I can not see any curve (for CYBR green) related to DNA copy increase and after finishing of the experiment it seems that DNA is not copied and not increased. do you know what is the problem? I should say that the annealing temperature of my primers in normal PCR is 59 C. that is why I just change this temperature with the device recommended time (20 sec). another recommended regulation of device is Denaturing temperature in 95 C (15 sec) and Elongation temperature in 72 (30 sec). Do you know what is the problem?
Thanks all.
1. I hope you have selected dye during plate set up. e.g. select SYBR as your target dye and also select reference dye (e.g. ROX) and if you have normalizer dye. You have to select dye for your controls, standards, unknowns, NTC.
https://www.youtube.com/watch?v=E8GzDgkuKGE
2. You have to standardize the parameter for denaturation, annealing and number of cycles according to your target amplification and it varies according to your primers set.
Do these primers amplify a product in a normal PCR reaction? Do your trouble-shooting for annealing temp and extension time using normal PCR, not in your qPCR.
Run out some of your qPCR products on an agarose gel to see if the issue is that you aren't getting any amplification OR if you are getting amplification, but didn't set up the machine to detect your SYBR dye.
Thanks a lot Dear Bhairavi Vajaria.
Yes, I have selected both SYBR and ROX dyes but there was not any option to specify each dye as a target or reference dye. Actually there is not any option in the instrument screen but there is in AriaMx software that I install in my PC. I don’t know why? And what should I do?
Also, I have selected only calibrator and unknown wells (for both the interest gene and housekeeping gene as a internal control). Is it wrong? Should I add standards and NTC?
Yes, I change the annealing temperature according to my primers actions.
Thanks so much Katie A Burnette.
Yes I have checked the thermal profile of this reaction by normal PCR. But somebody recommend me that apply recommended thermal setting according to master mix protocol. I did it and just changed its annealing temperature with my primer annealing temperature. Is it wrong strategy?
I also run the products on agarose gel. There was not any band.
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