We are running qPCR with SYBRGreen to amplify an inserted 12S ribosomal DNA region of a plasmid. When we ran this amplification for the first time (in duplicate), one replicate looked fine while the other presented with negative RFU values, shifting the entire axis (see qPCR 4-2-18 image). We thought this may have been due to problems with SYBR, so we repeated the experiment with a new tube of SYBR. While the replicates using the "problematic" SYBR were fine, the "new" SYBR presented results similar to those seen on 4/2 (see qPCR 4-3-18). Approximately 14 ng of plasmid/DNA were loaded for each sample.
Has anyone ever seen this problem where the RFUs are negative like this?
Thank you for any assistance you may be able to provide.
Your assay is fine. It's the software that makes a problem here: the background correction is not working well. One reason for this is that the curves have very low ct values, so there is not much data the sotware can use to estimate the background, takes a wrong part of the curve as background and so produces this problem.
I suggest to dilute your template 1:100, you can even use 1:1000 dilutions! This will shift the ct values by 6-7 (or 10-11) cycles, so they will still be below 20. This should solve your problem.
Your assay is fine. It's the software that makes a problem here: the background correction is not working well. One reason for this is that the curves have very low ct values, so there is not much data the sotware can use to estimate the background, takes a wrong part of the curve as background and so produces this problem.
I suggest to dilute your template 1:100, you can even use 1:1000 dilutions! This will shift the ct values by 6-7 (or 10-11) cycles, so they will still be below 20. This should solve your problem.
I'm completely agree with Dr. Jochen. Also, you may try the following qPCR Troubleshooting...
http://blog.biosearchtech.com/TheBiosearchTechBlog/bid/63622/qPCR-Troubleshooting
Regards
Thank you both for the assistance. Bio-Rad and both of you had the same suggestions. We re-ran the qPCR this morning with a 1:1000 dilution of the plasmid, and we no longer had the problem. Samples came up around 16 or 17 cycles. Thanks again.
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