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库存 :roche biotin RNA labeline mix
英文名 :roche biotin RNA labeline mix
CAS号 :11685597910
保质期 :生物素RNA标记混合物
供应商 :Roche
保存条件 :生物素RNA标记混合物
规格 :40 μL
roche biotin RNA labeline mix生物素RNA标记混合物
orm | solution |
usage | sufficient for 20 reactions (transcription) |
packaging | pkg of 40 μL |
mfr. no. | Roche |
10x concentrated solution with: 10mM each ATP, CTP, GTP, 6.5mM UTP, 3.5mM Biotin-16-UTP, pH 7.5
Principle
Biotin-labeled, single-stranded RNA probes of defined length are generated by in vitro transcription. Biotin-16-UTP is incorporated by SP6, T7, and T3 RNA polymerases at approximately every 20 to 25th nucleotide of the transcript under the conditions described below. The Biotin RNA Labeling Mix is specifically designed for the use with Roche SP6,T7, and T3 RNA Polymerases, which are supplied with an optimized transcription buffer.
Preparation Note
Assay Time: 135 minutes
Sample Materials
• Linearized plasmid DNA
The DNA to be transcribed is cloned into the polylinker site of an appropriate transcription vector which contains adjacent to the polylinker a promoter for SP6, T7 or T3 RNA polymerase. For the synthesis of ′run off′ transcripts the plasmid is linearized by a restriction enzyme. Restriction enzymes creating 5′-overhangs should be used; 3′ overhangs should be avoided. The linearized template DNA should be purified by phenolchloroform extraction and ethanol precipitation, to avoid RNase contamination. For ′run around′ transcription circular plasmid DNA is used.
• PCR product
PCR-fragments which contain RNA polymerase promoter sequences can also act as templates for transcription. Purification of the correct PCR fragment by gel electrophoresis prior to transcription is recommended.
Labeling Efficiency
A standard labeling reaction with 1μg linear template DNA and an RNA polymerase produces approx. 10μg of full-length, biotin-labeled RNA. In a spot test, a combination of anti-biotin-AP and the chemiluminescence substrate CSPD can detect as little as 0.3pg of the biotin-labeled RNA.
Quality
Function tested in combination with the DIG RNA Labeling Kit.
Specificity
Heat inactivation: Stop the reaction by adding 2 μl 0.2 M EDTA (pH 8.0).
一般描述
特异性
应用
特点和优势
10x 浓缩液包含: ATP、CTP、GTP各10mM,6.5mM UTP,3.5mM生物素-16-UTP,pH 7.5
质量
原理
制备说明
样品材料
- 线性化质粒DNA:待转录的DNA将会被克隆至含有SP6,T7或T3 RNA聚合酶启动子并相邻于多接头的合适转录载体的多接头位点。对于′流失′转录本的合成,质粒是通过限制性酶进行线性化的。应使用限制性酶生成的5′-突出;应避免3′突出。线性化模板DNA应采用酚氯仿萃取和乙醇沉淀法纯化,以避免RNase污染。为′完成′转录,使用了环状质粒DNA
- PCR产物:含有RNA聚合酶启动子序列的PCR片段,也可以作为转录模板。推荐在转录前通过凝胶电泳纯化正确的PCR片段。
- 标记效率:μ通过1μg线性模板DNA和RNA聚合酶进行标准标记反应,产生大约10μg全长生物素标记RNA。在斑点试验中,联用抗生物素-AP与化学发光底物CSPD可检测低至0.3pg的生物素标记RNA。
其他说明
上海研卉生物科技有限公司
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入驻年限:10年