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AF647切口平移标记试剂盒
AF647切口平移标记试剂盒的结构式
AF647的激发光谱和发射光谱
For general laboratory use.
Shipping: shipped on gel packs
Storage Conditions: store at -20 °C
avoid freeze/thaw cycles, store dark
Shelf Life: 12 months
Spectroscopic Properties: λexc 648 nm, λem 671 nm, ε 270.0 L mmol-1 cm-1 (Tris-HCl pH 7.5)
Description:
AF647切口平移标记试剂盒包含切口平移标记所需的所有试剂(除模板和纯化探针的材料外),是一种高效、易操作和快速的标记技术。
该试剂盒推荐用于 DNA 的直接酶标记。AF647 NT 标记混合物含有经过特别优化的 dUTP-XX-AF647,可通过使用 DNA 聚合酶 I 进行缺口翻译将其掺入 DNA 中。该荧光团具有出色的稳定性和量子产率,同时染料-dUTP 复合物的掺入率很高,因此是各种荧光应用的理想选择。
DNase I 能在低酶浓度下将随机分布的刻痕引入双链 DNA。聚合酶 I 的 5'→3' 外切酶活性可去除缺口 3' 侧的核苷酸,同时以 3'-OH 端部为引物合成部分新的互补链。在存在染料标记的 dUTP 的情况下,聚合酶 I 结合标记的 dUTP 而不是 dTTP。混合酶中均衡的聚合酶/核酸酶活性可确保生成高度标记的双链 DNA 片段。
生成的 DNA 适合用于 FISH、微阵列基因表达谱分析和其他核酸杂交检测。
保护荧光标记的 dUTP 免受光照,在弱光条件下进行实验操作。
Content:
Enzyme mix (red cap)
2 units/μl polymerase I, 0.02 units/μl Dnase I in storage buffer
NT labeling buffer (green cap)
10x conc.
AF647 NT labeling mix (purple cap)
0.5 mM dATP, 0.5 mM dCTP, 0.5 mM dGTP, 0.25 mM dTTP,
0.25 mM dUTP-XX-AF647, pH 7.5
Stop buffer (yellow cap)
0.5 M EDTA, pH 8.0
PCR-grade water (white cap)
Recommended NT assay:
Sample Material can be supercoiled or linearized plasmid DNA, cosmid or BAC DNA, whole or partial chromosomes or purified PCR products.
Prepare the following reaction mixture in a sterile vial.
20 μl Nick translation labeling assay
fill up to 20 μl | PCR-grade water | white cap |
2 μl | 10x NT labeling buffer | green cap |
2 μl | AF647 NT labeling mix |
purple cap |
1-1.5 μg | template DNA | - |
2 μl | Enzyme mix | red cap |
- Vortex the mix gently to assure homogeneity and centrifuge briefly to collect the reaction mixture at the bottom of the tube.
- Place the tube in a precooled thermomixer at 15 °C. An incubation of 90 min is recommended to generate DNA fragments in a size range between 200 and 500 bp.
- To control the length of the fragments load 2 μl of the assay on an agarose gel. Place the reaction tube at -20 °C while running the gel.
- To get smaller fragments add additional 2 μl of the Enzyme mix and extend the incubation at 15 °C.
- For final stopping the reaction add 5 μl of Stop buffer (yellow cap). Proceed to purification or store at -20 °C.
Purification of the probe:
To remove unincorporated nucleotides from the reaction mixture prior to its use in subsequent experiments one of the following procedures is recommended:
1. Purification by silica-gel membrane adsorption - PCR Purification Kit, Cat.-No. PP-201
The Jena Bioscience PCR Purification Kit provides a simple and efficient way to purify DNA fragments larger than 100 bp. The preparation is based on a silica-membrane technology for binding DNA in high-salt and elution in low-salt buffer. Please refer to the instruction manual.
2. Purification by Isopropanol precipitation
Add 1 μl glycogene (2 mg/ml), 2 μl sodium acetate (3 M) and 14 μl isopropanol to the reaction mixture and mix well but gently. Incubate on RT for 15 min and spin down at maximum speed at 4 °C for 30 min. Discard the supernatant and wash 2x with 70 % ethanol (spin down at maximum speed for 5 min).
3. Purification by Centrifugal Filter Units
Unincorporated nucleotides can be removed by centrifugation using centrifugal filter units. Select the filter unit by its cut-off for DNA fragments and follow the manufacturer's instructions.
Incorporation rate of the fluorophore:
The efficiency of DNA labeling can be estimated by calculating the ratio of incorporated fluorophores to the number of bases in the fragment (dye / base).
1. Measurement of the optical density: Measure the absorbance of the labeled DNA fragment at 260 nm (A260) and at the excitation maximum (λexc) for the dye (Adye).
2. Correction of the A260 reading: To obtain an accurate absorbance measurement for the nucleic acid, the contribution of the dye at 260 nm has to be corrected. Use the following equation:
Abase = A260 - (Adye x CF260)
Correction Factor for AF647: CF260 = 0.06
3. Calculation of the labeling rate: The dye to base ratio is given by:
dye / base = (Adye x εbase) / (Abase x εdye)
Extinction coefficients:
AF647: εdye = 270,000 cm-1 M-1
dsDNA: εbase = 6,600 cm-1 M-1
ssDNA: εbase = 8,900 cm-1 M-1
oligonucleotide: εbase = 10,000 cm-1 M-1
Example: A dye to base ratio of 0.05 corresponds to an incorporation of 10 dye-dUTP nucleotides into a DNA fragment containing 200 nucleotides or, respectively, into a 100 bp PCR fragment. If an equal distribution of dATP, dCTP, dGTP and dTTP in the DNA fragment can be assumed, 10 of the 50 existing dTTPs have been substituted by dye-dUTP resulting in a labeling rate of 20 %.
产品组分
内容 |
型号 |
规格 |
储存温度 |
AF647切口平移标记试剂盒 | PP305AF647S | 10次 | 室温 |
AF647切口平移标记试剂盒 | PP305AF647L | 50次 | 室温 |
操作手册 |
1 | 1 |
常温 |
注意事项
保存建议 | 厂家推荐蓝冰运输。当您收到产品后,按照说明书建议保存于-20°C。 |
FAQ
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