SYBR green I chloride | CAS# 2225748-05-8 MCE貨號HY-D1191
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MCE (MedChem Express) Fluorescent Dye -SYBR green I chloride
MCE (MedChemexpress) 總部位於美國新澤西，提供種類齊全的化學及生化研究用產品，包括創新的生命科學試劑，reference compounds (標準化合物)，APIs (原料藥) 及Natural compounds (天然化合物)以提供實驗室或科學研究之用，MedChemExpress(MCE)產品50000+ 特異性抑制劑、化合物庫、重組蛋白，專注於信號通路和疾病研究領域。全球文獻廣泛引用，質控體系嚴格，品質100%。官網連結 https://www.medchemexpress.com
SYBR green I chloride | CAS# 2225748-05-8 MCE貨號HY-D1191
SYBR Green I chloride is a highly sensitive fluorescent nucleic acid dye that binds specifically to the minor groove of double-stranded DNA or intercalates between base pairs.
產品介紹: MCE品牌提供的一系列化合物，SYBR Green I 氯化物 (SYBR Green I chloride) 是一種高靈敏度的螢光核酸染料 (fluorescent nucleic acid dye)，可特異性結合雙股 DNA 的小溝或嵌入鹼基對之間。 SYBR Green I 氯化物在未結合狀態下螢光較弱，結合後則發出明亮的螢光，並且優先結合大片段 DNA 和高 G+C 含量的 DNA。 SYBR Green I 氯化物適用於即時螢光定量 PCR 技術 (real-time PCR technology)；其螢光強度與擴增產物的數量和大小相關，無需額外的後處理，即可透過熔解曲線分析實現基因表現的精確定量和擴增子的區分。 SYBR Green I 氯化物廣泛應用於臨床前體外核酸檢測 ( in vitro nucleic acid detection)。
For research use only. We do not sell to patients.
SYBR green I chloride Chemical Structure
CAS No. : 2225748-05-8
1. Sample and Nucleic Acid Preparation
Cell treatment:
Splenic monocytes are isolated from mice and divided into a control group and a PMA-ionomycin stimulation group, followed by incubation at 37℃ with 5% CO2 for 5 h.
RNA extraction:
Cells are lysed with TRIZOL to extract total RNA; genomic DNA is removed with DNase I, RNA integrity is verified via denaturing gel electrophoresis, and RNA is quantified at 260 nm.
cDNA synthesis:
2 μg of RNA is annealed with 1 μg of oligo-dT; a reverse transcription system is added, and cDNA is synthesized through the procedure of 25℃ for 5 min → 37℃ for 60 min → 70℃ for 10 min.
2. Reaction System Preparation (50 μL total volume)
The system contains 0.5×SYBR Green I (10000× stock solution), PCR buffer (50 mM KCl, 20 mM Tris-HCl pH 8.3, 2.5 mM MgCl2, 0.2% glycerol, 0.2% DMSO), 0.2 mM forward and reverse primers, 0.2 mM dNTPs, 1.25 U AmpliTaq, and 5 μL of 1:5 diluted cDNA.
No-template controls (NTC) are set up, and all samples are run in at least 2 replicates.
3. Amplification Cycling Program
Pre-denaturation is performed at 95℃ for 30 s; 35 cycles of amplification are carried out (95℃ for 30 s → 60℃ for 30 s → 72℃ for 1 min → 85℃ for 5 s).
Fluorescence collection:
Fluorescence is collected at 85℃ during each cycle (a temperature higher than the Tm of primer dimers and lower than the Tm of specific amplicons).
4. Melting Curve Analysis
After amplification, the temperature is increased from 60℃ to 95℃ at a rate of 2℃/min, and fluorescence is detected every 15 s.
Target amplicons and non-specific signals from primer dimers are distinguished based on specific Tm peaks.
5. Quantification and Specificity Verification
Data analysis:
The baseline is set to cycles 1-5, with a threshold of 0.7; Ct values are read by software, absolute copy numbers are calculated using an external plasmid standard curve (109-104 copies); β-actin is used as an internal reference for normalization to calculate relative expression levels.
Specificity verification:
A single target band is confirmed via 2% agarose gel electrophoresis, which matches the specific peak in the melting curve.
Multiplex Real-Time PCR Based on Bacterial Samples[2]
1. Template Preparation
Strain culture: *Vibrio cholerae* is inoculated onto Columbia blood agar and cultured aerobically at 35°C for 24 h; *Legionella pneumophila* is inoculated onto BCYE agar and cultured aerobically at 35°C for 3 days.
DNA extraction: For *Vibrio cholerae*, 2-3 colonies are resuspended in 200 μL Instagene, and DNA is prepared following the kit instructions; for *Legionella pneumophila*, a bacterial suspension with a turbidity of 0.5 McFarland standard is prepared as the template.
2. Reaction System Preparation (20 μL total volume)
Homemade SYBR Green I (SG) premix:
1× PCR Buffer II, 4 mM MgCl2, 200 μM dNTP, primers (0.5 μM each of *Legionella* 16S rRNA primers, 0.1 μM each of mip primers; 0.5 μM each of singleplex mip primers), 1 U AmpliTaq Gold, SG at a final concentration of 0.2× or 0.4× (from 10000× stock solution), and 4 μL template DNA.
3. Amplification Cycling Program
For *Vibrio cholerae*: Pre-denaturation at 95°C for 10 min; 40 cycles of 94°C for 20 s → 60°C for 45 s → 72°C for 45 s.
For *Legionella pneumophila*: Pre-denaturation at 95°C for 10 min; 30 cycles of 94°C for 10 s → 50°C for 20 s → 72°C for 25 s.
Fluorescence collection: Performed at 72°C in the SYBR channel (excitation at 470 nm, detection at 585 nm) with gains set to 2 and 5.
4. Melting Curve Analysis
After amplification, the temperature is increased from 75°C to 99°C at a rate of 1°C per 60 s.
Software analysis: RotorGene V4.6.94 is used with digital filtration set to none, and the Tm value of amplicons is read.
5. Product Verification
1% agarose gel electrophoresis (with Gelstar dye added) is performed, and the melting curve is compared with the electrophoresis bands to verify the amplification of multiplex products.
6. Key Usage Points
In multiplex PCR, an SG concentration of **≥0.4×** is required for simultaneous detection of multiple amplicons; a concentration of 0.2× only detects products with high GC% and large fragment sizes.
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