I use end-point PCR cleanup product as a template for my Real time PCR standard curve studies (10-fold dilutions). My slope always ranges between -3.7 to -3.9, instead of an ideal 3.3. What could be the issue and how can i rectify it to obtain a -3.3 to -3.4 slope?
What is the highest concentration of serial dilution you used as template for your qPCR reaction? If it is undiluted or even 10 times diluted, that would be the issue of too much template!
Ali Javadmanesh The original was 1ng and 1:10 dilutions (7 dilutions) were carried out.
Thermo Fisher has a great website to help:
https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/real-time-pcr-learning-center/real-time-pcr-basics/real-time-pcr-troubleshooting-tool/gene-expression-quantitation-troubleshooting/poor-pcr-efficiency.html
First, see if you have any outliers at your lowest template concentration. If that doesn't fix the issue, then it's either PCR inhibitors (not likely using a cleaned PCR as template) or you simply need to design new primers. Not all primers are efficient enough to use for qPCR
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