Hello,
I'm at the stage of optimizing my qRT-PCR protocol. Can anyone tell me why my plot looks like this?
I'm using Taqman advanced microRNA assay and universal PCR mastermix no amperase UNG..
I checked the conc. of cDNA, it looks fine.
Anybody has any idea?
Hi Nur,
Did you give a quick spin before placing the samples n to the machine? A small drop on the wall can make a lot of differences.
No, you're not at the stage of optimizing. You're at the stage of getting it to work at all. At least one essential component of your process is not working. We can't tell from looking at the trace which one it might be. It could be anything. Try to isolate as many components as you can, and validate them individually.
For example: Run the DNA on a gel. Make sure the instrument is warming up and cooling with a thermometer. Detects a reference dye. Can you get an unrelated control amplification to work? etc...
amplification or detection is not working. New assays? You should check the machine with positive controls that are available in most kits. Check the channels for detection. Once checked, you can begin to check that your primers amplify correctly. Load in a agarose gel to see the band. If you see the band and there is still not amplification, redesign the taqman probe avoiding polymorphisms
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biomolecules
- Nucleic Acids
- RNA
- Small RNA
- microRNA