SYBR Green I is a dye with a green excitation wavelength that binds to the minor groove region of all dsDNA double helices. In the free state, SYBR Green I emits weak fluorescence, but once bound to double-stranded DNA, the fluorescence is greatly enhanced. Therefore, the fluorescence signal intensity of SYBR Green I is related to the amount of double-stranded DNA, and the amount of double-stranded DNA present in the PCR system can be detected based on the fluorescence signal. The maximum absorption wavelength of SYBR Green I is about 497nm, and the maximum emission wavelength is about 520nm.
The main purpose:
SYBR Green I is a highly sensitive DNA fluorescent dye, suitable for various electrophoretic analysis, and the operation is simple: no decolorization or washing is required. At least 20pg of DNA can be detected, which is 25-100 times higher than that of EB staining. When SYBR Green I combines with dsDNA, the fluorescence signal will be enhanced by 800-1000 times. The gel sample stained with SYBR Green I has strong fluorescence signal and low background signal. It can be applied to various electrophoresis analysis.
SYBR Green I is suitable for various gel electrophoresis methods: agarose gel, polyacrylamide gel electrophoresis, pulsed field gel electrophoresis, and capillary electrophoresis, etc.
SYBR Green I has a very high affinity with double-stranded DNA, so it can be used as pre-electrophoresis staining, and has no inhibitory effect on enzymes commonly used in molecular biology (such as: Taq enzyme, reverse transcriptase, endonuclease, T4 ligase, etc.) . In addition, compared with EB, SYBR Green I has greatly reduced mutagenic ability.
SYBR Green I nucleic acid gel stain (SYBR Green Nucleic Acid Gel Stains) is a highly sensitive fluorescent stain for the detection of double-stranded DNA and oligonucleotides in agarose gels and polyacrylamide gels . When the stain is combined with nucleic acid, the fluorescent signal of SYBR Green I nucleic acid gel stain will be significantly enhanced, so the gel stained with SYBR Green I nucleic acid gel stain shows a very good signal-to-noise ratio without background fluorescence. For best results, it is best to run the gel before staining it. SYBR GreenI Nucleic Acid Gel Stain is used for low-level PCR products, and is ideal for the detection of small amounts of DNA in apoptosis research and heteroduplex analysis. Can be applied to: DNA and RNA detection; SSCP and heteroduplex analysis.
main feature:
High sensitivity: at least 20pg of DNA can be detected, which is 25-100 times higher than that of EB staining.
High signal-to-noise ratio: strong sample fluorescence signal and low background signal.
Easy to use: No bleaching or rinsing required.
Wide range of applications: applicable to a variety of electrophoresis analysis.
Easy to use: It does not affect the action of other modifying enzymes.
Economical: The price is cheaper than silver staining.
Pre-staining method:
1. This method is suitable for agarose gel electrophoresis and PAGE gel electrophoresis
2. Preparation of working solution: Dilute 10000× SYBR Green Ⅰ by 100 times with electrophoresis buffer to obtain SYBR Green Ⅰ working solution. SYBR GreenⅠ working solution can be refrigerated at 2-8°C for more than one month.
3. Glue making: make glue according to the conventional method without any dye.
4. Sample staining: Add SYBR Green Ⅰ working solution and sample loading buffer to the analysis sample, and let it stand at room temperature for 10 minutes to fully combine SYBR Green Ⅰ with the DNA in the sample. The amount of SYBR Green I working solution added was 1/10 of the total sample amount.
5. DNA Marker staining: Mix 5 μL DNA Marker and 1 μL SYBR Green Ⅰ working solution, and let stand at room temperature for 5 minutes to fully combine SYBR Green Ⅰ with DNA.
6. Sample loading and electrophoresis: operate as usual.
7. Detection: detection with ABLUe visible light nucleic acid detection instrument
After dyeing method:
1. Perform electrophoresis according to conventional methods.
2. Dilute SYBR Green I Concentrate with a pH 7.0 – 8.5 buffer (such as TAE, TBE or TE) at a ratio of 10000:1 and mix well to make a staining solution.
3. Pour the staining solution into a suitable polypropylene container, place the gel in, and cover the container with aluminum foil, etc. to protect the dye from light. Shake and stain at room temperature for 10-30 minutes. The staining time depends on the concentration and thickness of the gel. The polyacrylamide gel was directly stained on the glass plate, and the prepared working solution was gently poured on the gel plate, so that the working solution evenly covered the entire gel plate, and stained for 30 minutes. Glass dishes must be pre-treated with a silanization solution (to avoid dye adsorption on the glass surface).
4. Observe with ABLUe. Blue light can pass through the glass. When observing polyacrylamide gel, you can directly put the glass plate holding the gel into the ablue for observation.
Precautions:
1. In the “SYBR Green Ⅰ pre-staining method”, the electrophoresis time should not exceed 2 hours, otherwise SYBR Green Ⅰ will separate from the DNA and produce diffuse bands.
2. SYBR Green I can be completely removed from double-stranded nucleic acid during routine alcohol precipitation.
3. If you want to perform Southern blots on gels stained with SYBR Green I, it is recommended to add 0.1%-0.3% SDS to the prehybridization and hybridization solutions.
4. Under UV light perspective, SYBR Green I conjugated to double-stranded DNA exhibits green fluorescence. If the gel contains single-stranded DNA the color will be orange instead of green.
5. SYBR Green has a certain affinity for glass and non-polypropylene materials. It is recommended to use polypropylene containers during dilution, storage, dyeing, etc.
Operation process:
1 Experimental method
Cystic tissue was quick-frozen in liquid nitrogen and stored in a -70°C refrigerator for later use. Real-time PCR method was used to detect the mRNA content of EMMPRIN.
1.1 Total RNA extraction from tissue:
(1) Take out the tissue from the liquid nitrogen, cut about 100mg of tissue pieces with treated scissors, add 1ml Trizol reagent, transfer to a homogenizer, place the homogenizer on ice water to grind fully, transfer the ground Transfer the homogenate to a 1.5ml centrifuge tube without RNase/DNase, and let stand at room temperature for 5 minutes.
(2) Add 0.2ml of chloroform, invert and mix 10 times to fully mix, let stand at room temperature for 2-5min, and centrifuge at 4°C at a centrifugal speed of 12000r/min for 10min;
(3) Carefully pipette the upper aqueous phase solution into another RNase/DNase-free 1.5ml centrifuge tube, carefully pipette the upper aqueous phase, do not touch or suck away the middle layer, otherwise centrifuge again and then pipette (can be pipette about 0.4-0.5 ml).
(4) Add an equal volume of isopropanol, invert and mix well, let stand at room temperature for 15 minutes, and then centrifuge at 4°C with a centrifugation speed of 12,000 r/min for 15 minutes. Carefully discard the supernatant, add 1ml of 75% ethanol (pre-cooled in DEPC water) along the tube wall, let it stand at room temperature for 1min, and centrifuge at 4°C at a speed of 7500r/min for 5min; pour off the supernatant carefully, and then Centrifuge at 4°C at a speed of 3500r/min for 1min; after centrifugation, use a 10μl pipette tip to carefully absorb the residual liquid, and finally dissolve the RNA with 30μl DEPC-H2O.
(5) The extracted total RNA was measured by UV spectrophotometer, and the ratio of extract concentration OD 260/280 was between 1.8-2.0.
1.2 Reverse transcription reaction:
Use 2 μg of total RNA extracted from the above cells to reverse-transcribe into cDNA in the following 20 μl reaction system. The composition of the reaction system is as follows: 5×RT-Buffer 4 μl, oligd(T) 2.5 μmol/L, dNTPs 5 mmol/L, RNase inhibitor Agent (RNAsin) 20U. First, pre-denaturation at 70°C for 5 minutes, then adding 200 U of reverse transcriptase MMLV, then incubating at 42°C for 1 hour, heating at 72°C for 10 minutes to inactivate reverse transcriptase.
1.3 Real-time PCR reaction:
1.3.1 Methodological confirmation According to the primer-related information PCR amplification, PCR products were analyzed by agarose gel electrophoresis and purified. Assuming that the purified DNA concentration was a×10x, it was serially diluted 7 times by 10 times, and the dilution concentration was Select the appropriate dilution for a×10x-1, a×10x-2, a×10x-3, a×10x-4, a×10x-5, a×10x-6, a×10x-7 as the standard template.
1.3.2 Take the above reverse transcription reaction product and carry out PCR reaction in 20μl reaction system.
PrimerSequence (5′to 3′) Length (bp)
GAPDH Forward primer 5′-GTCGGTGTGAACGGATTT-3’276
GAPDH Reverse primer5′-ACTCCACGACGTACTCAGC-3′
EMMPRINForward primer5′-TGGCCTTCACGTTCCTGAGT-3’215
EMMPRINReverse primer5′ – GTCATCTGCATCCACCGTGT-3′
The composition of the reaction system is as follows: 10×Buffer (2μl), dNTP 10 mmol/L (0.4μl), MgCl2 25mmol/L (1.6μl), 5U TaqDNA polymerase (0.3μl), template 2μl, upstream and downstream specific primers 0.25μl each 16.2 μl of deionized water.
Run according to the following thermal cycle parameters: GAPDH internal reference gene; pre-denaturation: 94×2min; denaturation: 94×2s, annealing 56×15s, extension 72×15s (lighting) for a total of 30 cycles. EMMPRIN gene; pre-denaturation: 94×2min; denaturation: 94×2s, annealing 58×15s, extension 72×12s (lighting), a total of 35 cycles. The Ct values of the target genes EMMPRIN and GAPDH were analyzed and calculated by SLAN software.
2 Experimental results
2.1 Agarose gel electrophoresis results of internal reference gene and target gene: PCR amplification products of 3 specimens were randomly selected for agarose gel electrophoresis, and there was a single band at the position of the theoretical fragment size and no miscellaneous bands and primer-dimers.
2.2 Reagent amplification efficiency verification: After quantification of the standard, the correlation coefficient between the Ct value of the internal reference gene GAPDH amplification detection and the logarithmic concentration value was 0.99996, and the correlation coefficient between the Ct value of the target gene EMMPRIN amplification detection and the logarithmic concentration value was 0.99969. Both the internal reference gene and the target gene have good amplification efficiencies (correlation coefficient >0.98). Data statistics can be performed by the ΔCt method.
