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View additional product information for ChromaTide™ Texas Red™-12-dUTP - FAQs (C7631)
19 product FAQs found
你可以尝试使用合适的基于离心柱的方法纯化ChromaTide标记的探针以去除未掺入的ChromaTide核苷酸。乙醇沉淀可能不能有效的去除未掺入的ChromaTide核苷酸，因此需要使用离心柱进行纯化。
•检查碱基和染料的比例，确定ChromaTide核苷酸掺入水平。由于荧光检测可能受标记不足、过度标记、仪器灵敏度或其它因素影响，所以碱基与染料的比例是更好的检测掺入效率的指标。
•ChromaTide核苷酸在酶促标记反应时可能没有很好的掺入。确保所使用的酶促方法与特定的荧光ChromaTide核苷酸兼容，因为一些方法可能并不是对所有应用都适合。您可能还需要进一步优化酶促掺入方法，如优化酶浓度、孵育时间、浓度及标记核苷酸与未标记核苷酸的比例。对于PCR，保真度低一些的聚合酶可以得到更高的掺入率；当然，使用PCR时，掺入率通常都较低。
•检查检测所用荧光滤光片，确保其与染料相兼容。你也可以在所用滤光片下检测一小滴没有稀释的染料，确保在没有与寡核苷酸结合前你能对染料单独成像。Alexa Fluor 647的发射荧光肉眼不可见，因此需要远红外成像系统进行检测。
不能，它们不是细胞通透的所以它们仅适用于体外掺入方法。荧光染料和磷酸基团电荷太高因而不能穿透完整细胞膜。不带磷酸基团的非荧光染料例如EdU, EU, 或BrdU可以用于活细胞核酸掺入研究。
碱基-染料比例可以通过测定核酸在260 nm的吸光值和染料在其最大吸收峰的吸光值而确定。使用相应染料和核酸的消光系数，您就可以通过Beer-Lambert定律计算出被标记核酸的碱基-染料比例。详细的说明可以在下列产品说明中找到：
酶促方法掺入ChromaTide dUTP (http://tools.thermofisher.com/content/sfs/manuals/td07604.pdf)
酶促方法掺入ChromaTide UTP(http://tools.thermofisher.com/content/sfs/manuals/td07605.pdf).
平均掺入率是大约每100-150碱基掺入一个染料分子，所以ChromaTide荧光标记核苷酸通常是我们提供的核酸标记方法里标记率最低的。
虽然我们有一个使用PCR进行掺入的实验方案，但是我们不建议使用PCR作为任何荧光或修饰核苷酸的最佳掺入方法。使用PCR时，掺入率非常低。我们曾经使用Taq聚合酶成功过；但是很多现在的DNA聚合酶保真度更高而且合成错误更少；因此，它们掺入修饰核苷酸的效果不是很好。下列文章对不同商品化的聚合酶对不同染料或修饰的核苷酸在PCR反应中掺入效率进行了比较：
Anderson JP, Angerer B, Loeb LA (2005) Incorporation of reporter-labeled nucleotides by DNA polymerases. Biotechniques 38:257–264.
能够将ChromaTide dUTP高效掺入DNA的方法有切口平移，随机引物标记，通过末端转移酶（Tdt）进行的末端标记，以及逆转录。ChromaTide UTP可通过体外转录掺入到RNA中。你可以在下面找到使用这些酶促反应的实验步骤。某些荧光标记的ChromaTide核苷酸可能不适用于某个特定的酶促方法，所以我们建议您在购买前参阅ChromaTide产品手册中的表1。一些ChromaTide产品已经停产，所以并不是所有列出的ChromaTide核苷酸目前都是可订购的：
ChromaTide标记核苷酸产品手册(http://tools.thermofisher.com/content/sfs/manuals/mp07603.pdf)
酶促方法掺入ChromaTide dUTP(http://tools.thermofisher.com/content/sfs/manuals/td07604.pdf)
酶促方法掺入ChromaTide UTP(http://tools.thermofisher.com/content/sfs/manuals/td07605.pdf)
ChromaTide dUTP和 UTP核苷酸是通过一个独特的烷基侧链接头对尿嘧啶C-5位进行修饰的。这个接头可以增加核苷酸和染料之间的间隔从而减少他们之间的相互作用。这些核苷酸中的一些还包含一个额外的spacer，从而进一步将核酸和标记基团隔开。产品名称中的数字，例如fluorescein-12-dUTP中的“12”，指的是spacer的净原子数长度。
ChromaTide染料标记的dUTP和UTP核苷酸可以被用来合成带有标记的核酸探针而无需接触危险而且昂贵的同位素标记的核苷酸。您可以使用标准分子生物学技术将这些带标记的核苷酸掺入探针，然后用带标记的探针进行原位杂交、基因芯片或印迹分析。ChromaTide染料标记的核苷酸有多种荧光颜色可供选择，便于进行多色分析。
You can try to purify the ChromaTide labeled probe with an appropriate spin column-based method to remove unincorporated ChromaTide nucleotides. Ethanol precipitation may not efficiently remove the unincorporated ChromaTide nucleotides, so a spin column will need to be used.
- Check the base-to-dye ratio to determine the level of incorporation of the ChromaTide nucleotides. Since fluorescent detection may be affected by underlabeling, overlabeling, instrument sensitivity, or other factors, the base-to-dye ratio is a better indicator of incorporation efficiency.
- ChromaTide nucleotides may not have been incorporated well in the enzymatic labeling reaction. Make sure that the enzymatic method used is compatible with the particular fluorescent ChromaTide nucleotide, since some methods may not be appropriate for all applications. You may also need to further optimize the enzymatic incorporation method, for example by optimizing enzyme concentration, incubation time, concentration, and ratio of labeled and unlabeled nucleotides. For PCR, a lower fidelity polymerase may give higher incorporation rates; however, incorporation rates will be generally low using PCR.
- Check the fluorescent filter used for detection to make sure it is compatible with the dye. You can also test a small drop of the undiluted fluorescent ChromaTide nucleotide in your filter to make sure you can image the dye alone before it is conjugated to the oligonucleotide. The fluorescence emission of Alexa Fluor 647 is not visible by eye and will require a far-red imaging system for detection.
No, they are not cell permeant so they are only suitable for in vitro incorporation methods. The fluorescent dyes and phosphate groups are too highly charged to allow the nucleotides to penetrate the membrane of an intact cell. Nonfluorescent nucleosides without phosphates such as EdU, EU, or BrdU can be used for live cell nucleic acid incorporation studies.
The base-to-dye ratio is determined by measuring the absorbance of the nucleic acid at 260 nm and the absorbance of the dye at its absorbance maximum. Using the extinction coefficients for the appropriate dye and nucleic acid, you can then calculate the base-to-dye ratio for the labeled nucleic acid using the Beer-Lambert law. Detailed instructions can be found in these product manuals: (http://tools.thermofisher.com/content/sfs/manuals/td07604.pdf, http://tools.thermofisher.com/content/sfs/manuals/td07605.pdf).
The average incorporation is one dye for every 100-150 bases, so the ChromaTide fluorescently labeled nucleotides typically produce the lowest labeling rates of the nucleic acid labeling methods we offer.
Although we have a protocol for incorporation using PCR, we do not recommend PCR as the best method for incorporation of any of our fluorescent or modified nucleotides. With PCR, incorporation rates are very low. We had some success with Taq polymerases in the past; however, many of the modern DNA polymerases are higher fidelity; as a result, they do not incorporate modified nucleotides very well. This paper below provides a comparison of commercially available polymerases and PCR incorporation efficiency related to type of dye or modification:Anderson JP, Angerer B, Loeb LA (2005) Incorporation of reporter-labeled nucleotides by DNA polymerases. Biotechniques 38:257–264.
ChromaTide dUTPs are most efficiently incorporated into DNA using nick translation, random primer labeling, end-labeling with terminal deoxynucleotidyl transferase (TdT), and reverse transcription. ChromaTide UTPs can be incorporated into RNA using in vitro transcription. You can find example protocols using these enzymatic reactions below. Specific fluorescent ChromaTide nucleotides may not be appropriate for certain enzymatic methods, so we recommend consulting Table 1 in the ChromaTide product manual prior to purchase. Some ChromaTide products have been discontinued, so not all of the listed ChromaTide nucleotides may be currently available (http://tools.thermofisher.com/content/sfs/manuals/mp07603.pdf, http://tools.thermofisher.com/content/sfs/manuals/td07604.pdf, http://tools.thermofisher.com/content/sfs/manuals/td07605.pdf).
The ChromaTide dUTP and UTP nucleotides are modified at the C-5 position of uridine via a unique alkynylamino linker, which provides a spacer between the nucleotide and the dye to reduce interactions between them. Several of these nucleotides also contain an additional spacer, further separating the label from the nucleic acid. The number in the product name, e.g., the “12” in fluorescein-12-dUTP, indicates the net length of the spacer, in atoms.
ChromaTide dye-labeled dUTP and UTP nucleotides can be used to synthesize labeled nucleic acid probes without the need for hazardous and expensive radioisotope-labeled nucleotides. You can incorporate these nucleotides using standard molecular biology techniques, and then use the labeled probes for in situ hybridization, microarrays, or blotting. ChromaTide dye-labeled nucleotides are available with different fluorescent colors to facilitate multicolor analysis.
You will need to confirm with the manufacturer of the restriction enzyme you are using to find out whether or not it can recognize dUTP-containing DNA.
Find additional tips, troubleshooting help, and resources within our PCR and cDNA Synthesis Support Center.