I am using qPCR to amplify Shigella ipaH gene. This assay worked well in out lab previously, but now I cannot get good standard curve. My std. curve range is 10^6 to 10 gene copies, and 10^6 std. amplifies at Ct 18 as previously. Now there is a large gap between 10^6 and 10^5 Cts ( 10^5 amplifies at Ct 26). I changed all reagents and extracted new plasmid DNA, still I have the same problem. Any suggestions or recommendations?
There are several things why there is difference but if your ultimate goal is to calculate the PCR efficiency. Try using LinReg PCR software which is free and see if there is any difference between your previous and current results. You can also use this method to bypass the need for standard curve itself as this software can give you PCR efficiency based on each reaction in the plate and thus better represents the actual PCR efficiency than calculating it from standard curve... Check out papers mentioned on their website.
Here is a link to the home page
http://www.hartfaalcentrum.nl/index.php?main=files&sub=LinRegPCR
I hope this help you
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques