Anyone who has used SYBR Green in conventional PCR do let me know how gel was stained and quantity of SYBR Green used.
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SYBR Green® is a fluorescent dye that binds to double stranded DNA with great specificity. It is widely used in molecular biology research to quantify and visualize double stranded DNA. SYBR Green® is a synthetic dye, which was introduced in the early 1990s by Molecular Probes®, Inc., a subsidiary of Invitrogen&trade Corporation. It is an asymmetrical cyanine dye, cyanines being a family of synthetic organic dyes that are used in photography, CD-R and DVD-R media, and in a variety of molecular biology research applications.
Several features make SYBR Green® particularly useful as a nucleic acid stain. It is stable over a range of temperatures, which means that it can be used to monitor biochemical reactions that take place at high and low temperatures. It binds non-covalently to the surface of DNA, which is means that it will not interfere with the activity of most nucleases and DNA polymerases. It also binds to nucleic acids over a dynamic linear range of up to four orders of magnitude, so it can give an accurate quantification of DNA over a wide range of concentrations.
The most common laboratory uses of SYBR Green® are to detect double stranded DNA in real time polymerase chain reaction (PCR) and in acrylamide gels. PCR is a technique to amplify a few copies of DNA through repeated cycles of DNA replication. Real time PCR measures the amplification as it takes place, and since SYBR Green® binds to double stranded DNA, measuring the fluorescence emission of the DNA-dye complex allows the researcher to quantify the amount of DNA being produced. As PCR cycles progress, there is an increase in fluorescence proportionate to the amount of PCR product produced.
Acrylamide gels are ubiquitous in molecular biology labs, as they serve the commonly needed purpose of separating DNA fragments by size. SYBR Green® allows the researcher to visualize DNA in an acrylamide gel by shining light of the appropriate wavelength on it. Other uses of SYBR Green® include detection of nucleic acids in solution, in biochip applications, and in flow cytometry. The DNA-dye complex absorbs blue light at 488 nm and emits green light at 522 nm.
SYBR Green® is very sensitive; it is 25 times more sensitive than ethidium bromide, another commonly used dye for visualizing DNA. The high affinity of SYBR Green® for double stranded DNA makes it useful for detecting samples of DNA with low copy number. It preferentially binds double stranded DNA, but it can also bind single stranded DNA with reduced fluorescence. As with any DNA binding molecule, SYBR Green® can cause mutations and is a possible carcinogen. It is, however, safer to work with and easier to dispose of than ethidium bromide.
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