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For general laboratory use.
Shipping: shipped on gel packs
Storage Conditions: store at -20 °C
avoid freeze/thaw cyclesShelf Life: 12 months
Description:
生物素16切口平移(NT)标记试剂盒包含所有所需试剂（除模板和探针纯化材料外），用于基于NT的DNA生物素标记，提供一种高效、易操作且快速的标记技术。其标记原理与生物素-NT混合物（罗氏）类似。
NT标记基于聚合酶I和DNase I的反向活性。低浓度的DNase I能够在双链DNA上引入随机分布的切口。聚合酶I的5'→3'外切核酸酶活性从切口的3'侧移除核苷酸，同时以3'-OH末端为引物合成部分新的互补链。在标记dUTP存在的情况下，聚合酶I会掺入标记dUTP而不是dTTP。
BIO16 NT标记混合物（BIO16 NT labeling mix）含有优化的生物素标记dUTP，可通过NT使用DNA聚合酶I将其掺入DNA中。生物素-16-dUTP作为天然对应物dTTP的替代物被酶法掺入DNA。通过连接到尿嘧啶C5位的16原子接头确保了最佳底物特性、从而保证标记效率和生物素部分的有效检测。
该酶混合物中平衡的聚合酶/核酸酶活性，可确保生成高度标记的双链DNA片段。
所得生物素标记DNA适用于荧光原位杂交（FISH）及其他核酸杂交测定。
Content:
Enzyme mix (red cap)
2 units/μl Polymerase I, 0.02 units/μl DNase I in storage buffer10x NT labeling buffer (green cap)
10x conc.10x BIO16 NT labeling mix (purple cap)
0.5 mM dATP, 0.5 mM dCTP, 0.5 mM dGTP, 0.34 mM dTTP, 0.16 mM Biotin-16-dUTP, pH 7.5Stop buffer (yellow cap)
0.5 M EDTA, pH 8.0PCR-grade water (white cap)
Recommended NT assay (General Protocol):Sample material can be supercoiled or linearized plasmid DNA, cosmid or BAC DNA, whole or partial chromosomes or purified PCR products. PCR products should be purified by silica membrane adsorption to remove unincorporated dNTPs (e.g. PCR Purification Kit, #PP-201).
Prepare the following reaction mixture in a sterile vial.
20 μl Nick translation labeling assay
2 μl 10x NT labeling buffer green cap 2 μl 10x BIO16 NT labeling mix purple cap 1-1.5 μg template DNA - 2 μl 10x Enzyme mix red cap Fill up to 20 μl PCR-grade water white 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 2μl of the assay on an agarose gel. Place the reaction tube at -20°C while running the gel.
- To get smaller fragments extend the incubation at 15°C.
- For final stopping of the reaction add 5μl of Stop buffer (yellow cap). Proceed to purification or store at -20°C.
Purification of the probe:
Some downstream applications require the removal of unincorporated dNTPs (labelled and unlabelled). 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, #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 1μl 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. -
产品组分
内容 型号
规格 储存温度
生物素16切口平移(NT)标记试剂盒
CNBIO16-01 10次x20ul
-20°C 生物素16切口平移(NT)标记试剂盒
CNBIO16-02 75次x20ul
-20°C 操作手册
1 1 常温
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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。 -
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