Is it possible to cut a band from an agarose gel and purify it for subsequently creating a stock solution for qPCR? I use "gelgreen nucleic acid gel stain" (Biotium), for the agarose gel. Now I'm not sure if gelgreen can interact with sybre green during the qPCR reaction.
It is completely irrelevant if your dye "interacts" with SYBR Green ("interact" would mean that it shows a fluorescence at similar wavelength in the precence of dsDNA and light used to excite SYBR Green). It is only relevant that the fluorescence is low (undetectable) in the absence of dsDNA. The company states that it stains dsDNA 5x better than ssDNA, what is not so good for use in qPCR (SYBR Green is about 20x more sensitive for dsDNA). But if this is a problem depends on the final concentration of gelgreen in the qPCR mix. I would simply test it.
What could cause problems is a possible inhibitory effect on the DNA-polymerase. This effect is known to be very low for SYBR Green, but I habe no infor about gelgreen. One could ask the company. And also simply test it.
You could use SYBR Green directly to stain the gels (it was actually developed as a gel stain and was later used for qPCR, too). It should behave pretty much similar to gelgreen. That would avoid all your problems.
Why do you need to cut the band out of the gel? Most folks either just use a clean-up kit to remove unused primers and dilute DNA or clone their gene of interest into a plasmid to make their standard curve for qPCR.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids