- 产品描述
- 产品组分
- 文件资源
- 营销中心
- 注意事项
- FAQ
- 文献追踪
-
荧光素切口平移(NT)标记试剂盒的结构式
FAMX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 darkShelf Life: 12 months
Spectroscopic Properties: λexc 492 nm, λem 517 nm, ε 83.0 L mmol-1 cm-1 (Tris-HCl pH 7.5)
Description:
Fluorescein Nick Translation Labeling Kit contains all reagents (except template and materials for purification of the probe) required for nick translation labeling providing a highly efficient, easy-to-perform and rapid labeling technology.
The kit is recommended for direct enzymatic labeling of DNA. The Atto488 NT labeling mix contains specially optimized Fluorescein-12-dUTP for incorporation into DNA by nick translation using DNA Polymerase I. The excellent stability and quantum yield of the fluorophore combined with a high incorporation rate of the dye-dUTP complex makes it the ideal choice for a broad range of fluorescence applications.
Nick translation labeling is based on the reverse activities of Polymerase I and DNase I. DNase I is able to introduce randomly distributed nicks to double stranded DNA at low enzyme concentrations. The 5'→3' exonuclease activitiy of Polymerase I removes nucleotides from the 3' side of the nick while synthesizing a partial new complementary strand using the 3'-OH termini as primer. In presence of dye-labeled dUTP Polymerase I incorporates labeled dUTP instead of dTTP. The well balanced polymerase / nuclease activities of the enzyme mix ensure the generation of highly labeled double stranded DNA fragments.
The resultant DNA is suited for application in FISH, microarray gene expression profiling and other nucleic acid hybridization assays.
Protect fluorescent labeled dUTP from light and carry out experimental procedures in low light conditions.Content:
Enzyme mix (red cap)
2 units/μl polymerase I, 0.02 units/μl Dnase I in storage bufferNT labeling buffer (green cap)
10x conc.Fluorescein NT labeling mix (purple cap)
0.5 mM dATP, 0.5 mM dCTP, 0.5 mM dGTP, 0.25 mM dTTP,
0.25 mM Fluorescein-12-dUTP, pH 7.5Stop buffer (yellow cap)
0.5 M EDTA, pH 8.0PCR-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 assayfill up to 20 μl PCR-grade water white cap 2 μl 10x NT labeling buffer green cap 2 μl Fluorescein NT
labeling mixpurple 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 Fluorescein: CF260 = 0.32
3. Calculation of the labeling rate: The dye to base ratio is given by:
dye / base = (Adye x εbase) / (Abase x εdye)
Extinction coefficients:
Fluorescein: εdye = 83,000 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
荧光素切口平移(NT)标记试剂盒
PP305FAMX-01 10次
室温
荧光素切口平移(NT)标记试剂盒
PP305FAMX-02 50次
室温
操作手册
1 1 常温
-
营销中心
-
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
-
Natural repeated backcrosses lead to triploidy and tetraploidy in parthenogenetic butterfly lizards ( Leiolepis : Agamidae)
Eduard Galoyan, Roman Nazarov, ..., Lukáš Kratochvíl
Scientific Reports | 2025 Jan 24 | 39856096
Karyotype stasis but species-specific repetitive DNA patterns in Anguis lizards (Anguidae), in the evolutionary framework of Anguiformes
Marie Altmanová, Marie Doležálková-Kaštánková, ..., Vašek Gvoždík
Europe PMC | 2022 Dec 29
Sex chromosome differentiation via changes in the Y chromosome repeat landscape in African annual killifishes Nothobranchius furzeri and N. kadleci.
Jana Štundlová, Monika Hospodářská, ..., Alexandr Sember
Chromosome research : an international jou… | 2022 Dec 23 | 36208359
Uniparental Genome Elimination in Australian Carp Gudgeons
Zuzana Majtánová, Dmitrij Dedukh, ..., Alexander Suh
Genome Biology and Evolution | 2021 Jun 01 | 33591327
猜你喜欢
在线留言
如果您对我们的产品感兴趣，请留下您的信息，我们将尽快与您联系，谢谢！
艾维缔官网
艾德官网
B站IVDSHOW
抖音军哥聊表观
视频号艾维缔
小红书艾维缔
快手表观盒子
表观遗传学
联系我们
