Search
Search
View additional product information for ARES™ Alexa Fluor™ 488 DNA Labeling Kit - FAQs (A21665)
14 product FAQs found
不同批次制备的RNA肯定会得到不同的结果。大多数时候，mRNA是被显著降解的。酶促法掺入氨基酰dUTP（AA-dUTP）的效率在不同实验之间应该是相同的。如果有不同，则引起不同的原因应该是RNA或是方法本身。AA-dUTP的掺入和染料-核苷酸的偶联没什么不同，并且应该更高效和均一。
这里有几个建议：
1) cDNA可能在标记之前丢失了。在使用乙醇沉淀cDNA之前，可以向其中加入1 µL，内含10–20 µg的糖原（分子生物学级别）。
2) 确保加入的盐是醋酸钠而非醋酸铵。在cDNA沉淀之后，将其重悬在5 µL水中。
3) 如果生成的是长链cDNA，那么将样本加热变性将会有帮助。将样本在95°C 加热5分钟然后在冰上放置数分钟。然后在标记前将其离心数分钟。
4) 在进行标记反应时试管应当处于室温。向试管内加入3 µL缓冲液。然后加入染料并剧烈震荡混匀至少15秒。
很不幸的是，Alexa Fluor 633由于其化学结构而不能标记核酸。更重要的是，Alexa Fluor 633标记的DNA探针在核酸杂交实验中不能形成稳定的杂交。
ARES标记的寡核苷酸在95°C时5分钟应该没太大影响。
ARES标记的探针可以在任意缓冲液、TE、福尔马林、杂交缓冲液或乙醇中长期保存。我们建议使用您的常规存储条件保存探针，只需保持避光。ARES偶联物是非常稳定的。
在染料碱基比相同时，Alexa Fluor染料在高标记比时表现出更高的荧光强度和较低的自淬灭效应。
Alexa Fluor染料标记的cDNA用于基因芯片或FISH（荧光原位杂交）应用时，其染料-碱基比在1:12到1:35之间是合适的范围。
ARES Alexa Fluor DNA标记试剂盒提供了一种灵活的两步法对DNA进行Alexa Fluor染料标记。在第一步中，一个氨基修饰的核苷酸通过酶促标记方法被掺入到DNA中。在第二步中，氨基修饰的DNA被具有氨基反应活性的Alexa Fluor染料通过化学标记方法进行标记。标记后的探针可以用于：
•荧光原位杂交（FISH）。参阅Buster DW, Daniel SG, Nguyen HQ, et al. (2013) SCFSlimb ubiquitin ligase suppresses condensin II–mediated nuclear reorganization by degrading Cap-H2, J Cell Biol 201(1):49–63.
•基因芯片技术。参阅 Shin HH, Hwang BH, Seo JH et al. (2014) Specific discrimination of three pathogenic Salmonella enterica subsp. enterica serotypes by carB-based oligonucleotide microarray. Appl Environ Microbiol 80(1):366–373.
Different preparations of RNA will certainly give different results. Most of the time, the mRNA is significantly degraded. The enzymatic incorporation of aminoallyl-dUTP (AA-dUTP) should not differ from reaction to reaction. If there are differences, it has to be due to the RNA or the method. AA-dUTP incorporation is no different than that of a dye-nucleotide conjugate, and should be more efficient and uniform. Here are a couple of suggestions:
1) cDNA may have been lost prior to labeling. Add 1 µL of glycogen (molecular biology grade), containing 10-20 µg, to the cDNA before precipitating it with ethanol.
2) Make sure to add sodium acetate as the salt and not ammonium acetate. After pelleting the cDNA, resuspend it in 5 µL water.
3) If generating long cDNAs, it will help to heat-denature the sample. Heat it at 95°C for 5 minutes and then put it on ice for a few minutes. Then centrifuge it for a few minutes just prior to the labeling reaction.
4) You want the tube to be at room temperature for the labeling reaction. Add the 3 µL of buffer and mix it in. Then add the dye and vortex it vigorously for at least 15 seconds.
Unfortunately, Alexa Fluor 633 does not label nucleic acids well because of the dye's chemical structure. Furthermore, DNA probes labeled with Alexa Fluor 633 do not form stable hybrids in nucleic acid hybridization assays.
An ARES-labeled oligonucleotide should survive at 95°C for 5 minutes.
Long-term storage for the ARES-labeled probes can be done in just about any kind of buffer, TE, formamide, hybridization buffer, or ethanol. We suggest using your normal storage conditions as long as you protect the probes from light. ARES conjugates are very stable.
At the same dye-to-base ratio, Alexa Fluor dyes exhibit higher intensity and reduced self-quenching at higher labeling ratios.
A dye-to-base ratio of 1:12 to 1:35 is a suitable range for Alexa Fluor dye labeling of cDNA for microarray and FISH (fluorescence in situ hybridization) applications.
The ARES Alexa Fluor DNA Labeling Kits provides a versatile, two-step method for labeling DNA with our Alexa Fluor dyes. In the first step, an amine-modified nucleotide is incorporated into DNA using enzymatic labeling methods. In the second step, the amine-modified DNA is chemically labeled using our amine-reactive Alexa Fluor dyes. The labeled probes can be used for:
- Fluorescence in situ hybridization (FISH). See Buster DW, Daniel SG, Nguyen HQ, et al. (2013) SCFSlimb ubiquitin ligase suppresses condensin II–mediated nuclear reorganization by degrading Cap-H2, J Cell Biol 201(1):49-63.
- Microarray techniques. See Shin HH, Hwang BH, Seo JH et al. (2014) Specific discrimination of three pathogenic Salmonella enterica subsp. enterica serotypes by carB-based oligonucleotide microarray. Appl Environ Microbiol 80(1):366-373.