The RNA was treated with DNAse. The quantification was good.
Hi Ashley,
We need a bit more information. I assume you treated the sample with DNAse before procedeeing to make cDNA and the subsequent PCR with SYBRgreen. Is this correct or did you do something else?
Looking forward to hear from you,
Ali
Hi
I treated the RNA with DNAse and then I used the Kapa One step RTpcr protocol.
All right. This neans that you don't have a PCR product. My suggestion would be to repeat the experiment as follow. Do the same experiment with three different conditions. First add a negative control, meaning use water instead of the RNA. Second with the same RNA and third if you have another kind of RNA use that as a positive control.
I would like to see what kind of results would you get under these conditions.
Succes
Try to use any of the housekeeping genes as controls. Check Tm of you primers and design your experiment accordingly.
Hi Ashley,
first and foremost I would load the content of your Q-PCR wells in an agarose gel (if you didn't already). This would allow you to directly check what happened during the amplification...a sharp band at the expected size? a horrible smear (which btw would fit with the results of the melting curve)? something in between?
How did you inactivate the DNAse?
Hi I desperately needed Percision melting curve analysis for Mutation analsysis,any one now the alternate software to analyse the murtaion analysis of the data generated by Biorad Cfx Connect PLZ if you have percision melting curve software please email me [email protected]
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- RNA