When I perform a real-time PCR experiment (with for instance SYBR Green or EvaGreen), would you also stain the gel (to check the amplicon lenght) with this dye or is the presence of the dye from the real-time reaction sufficient?
Hi, just to add basic information, when you are performing SYBR green or Eva Green for Realtime and not the Taqman so you must know the science behind it, SYBR green only fluoresce when it binds to ds product but when we run then the melt curve analysis at the end of realtime pcr cycles to differentiate between real product, dimers or unspecific products in this case amplicons become ss again by high temperature and dye (SYBR green) releases and fluorescent drops, so my reaction mixture after the melt curve again have ss templates and floating dye without any fluorescence and when u want to confirm this product over the gel so definitely you need to stain your samples as SYBR green wont fluoresce now bcoz not in the binding state with your product, and also you always need to load your desired Ladder marker on gel to compare the bands lengths and we all know that this ladder is always stained and usually it is also preferred that the same stain of the ladder to be used to color your samples loading into the gel like bromophenolblue etc...so now you can visualize them only.
Most of the modern real time pcr experiments do not require gel electrophoresis. So the dyes used in your experiments will be enough for the software to calculate the targetted gene's expression. There is a substantial amount of literature online to help you choose the best method. From my experience absolute quantification works better
In principal you wouldn't need any dye if you already used it during the realtime PCR, however, the realtime PCR instruments are calibrated for much lower concentrations of the dye so you might not see it under the agarose gel. To be safe, I suggest using dye for the gel. Even if it turns out to be in excess it won't hurt.
I only want to check the amplicon lenght, without further quantification, to check if the primer design was correct. So I agree that an excess of dye would not hurt (it would only require an extra step, which might be avoided when the amount of dye in the amplicon would already be enough). However, realt-time PCR is indeed more sensitive than gel detection (especially if it turns out I have DNA of different sizes and therefore more than one band on the gel).
You may see the following link for options at chapter 12.
Book Labtop for Microbiology Laboratory
Are you also doing a post-cycling melt-curve to show the specificity of your primers? I think you could tell roughly the size from the Tm of the amplicon.
Presumably the amplicons generated during the cycling are already saturated with SYBR molecules so I am unsure what adding more SYBR will do. You may want to run quite a lot of the product because qPCR amplicons are typically below 200bp so you may need to add a lot of DNA to be above the detection limit (~50picogram?). This will also depend on what kind of gel-doc set up you have, i.e. how sensitive the camera is.
Hi, just to add basic information, when you are performing SYBR green or Eva Green for Realtime and not the Taqman so you must know the science behind it, SYBR green only fluoresce when it binds to ds product but when we run then the melt curve analysis at the end of realtime pcr cycles to differentiate between real product, dimers or unspecific products in this case amplicons become ss again by high temperature and dye (SYBR green) releases and fluorescent drops, so my reaction mixture after the melt curve again have ss templates and floating dye without any fluorescence and when u want to confirm this product over the gel so definitely you need to stain your samples as SYBR green wont fluoresce now bcoz not in the binding state with your product, and also you always need to load your desired Ladder marker on gel to compare the bands lengths and we all know that this ladder is always stained and usually it is also preferred that the same stain of the ladder to be used to color your samples loading into the gel like bromophenolblue etc...so now you can visualize them only.
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