I am working on a CFX 96 machine to determine reaction efficiency of my gene of interest.
After performing a serial dilution of my cDNA template, I was able to find great reaction efficiencies for 3/4 genes (102.5%).
One gene seemed to have odd results and when I reperformed the serial dilution I again found a similar pattern on the standard curve.
Sample PER B x10: 6 1:10 serial dilutions
Sample PER B x5: : 6 1:5 dilutions
Reaction:
10uL SyBER Supermix
7uL NF Water
1uL of each forward and reverse primer 5uM (primer concentration tested during a gradient with good product)
1uL of Stock cDNA produced from Trizol RNA extraction of animal brain 255ng/uL diluted as stated above
I have run NRTs with this sample and can rule out gDNA contamination
I have attached two screenshots of my standard curves
Is there any explanation for why I am getting such odd Cqs (~26) even after 6 serial dilutions?
If there is any more information I can provide please let me know
Ryan Scanlon How is the quality and quantity of cDNA?How is your NTC? Try with more then 6 point of the dilution.
Hi Ryan,
this seems to be strange. Have you tested the cDNA-dilution buffer for any contamination with your target?
Best,
Norman
Contamination seems the most likely culprit. Probably something that remains relatively constant between wells, since you see no dilution effect (so for example, your water, or your primers)
PCR produces massive amounts of specific templates, and if you're not incredibly careful, it's quite easy for a tiny droplet of completed reaction to accidentally contaminate a nearby stock. And a tiny droplet could contain trillions of templates. You'll see no effect with any other PCR, but the PCR for that specific template will give you something just like this.
Try repeating with fresh primer stocks and fresh water. And fresh master mix, too. Never hurts to replace everything.
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