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上海圻明生物
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羊肺炎链球菌探针法PCR检测试剂盒上海圻明生物优势供应。更多产品资料欢迎免费咨询。
One of the many important uses of PCR technology is that it can be used to label DNA probes with high specific activity. PCR technology has high specificity, and can synthesize probe DNA fragments in quantities within 1~2h if [α-32P]dNTP or other markers are added to the substrate
dNTPs, the probe DNA can be well labeled during DNA synthesis, and the incorporation rate of the marker can be as high as 70%~80%. Therefore, PCR labeling technology is particularly suitable for large-scale detection and non-radiolabeling. The disadvantage of this method is that a specific pair of PCR primers is synthesized.
Labeling can also be achieved by using small fragments prepared from probe DNA as primers.
Solution preparation
1. Prepare a stock solution
Unless otherwise stated, all unused stock solutions should be divided into disposable aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.
1.1* Acid Stock Solution (125X):
Add 20 μL DMSO to *ate (component B) to make a 125X* acid stock solution.
2. Prepare standard solutions
*Salt standard solution
Add 50 μL of 1 mM KH2PO4 (Component C) to 950 μL of deionized water or enzyme reaction buffer to give a 50 μM * saline standard solution (PS7). A 50 μM * saline standard solution (PS7) was taken and serially diluted 1:2 to obtain a serially diluted phosphate standard with deionized water or enzyme reaction buffer.
3. Prepare a working solution
Add 20 μL of 125X* stock solution to 2.5 mL of sterile H2O and mix well to make a working solution of *salt. Avoid potential Pi contamination. Note: Avoid direct exposure of *salts (component B) to light. Due to the high sensitivity of this assay to Pi, it is extremely important to use Pi-free labware and reagents.
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文献和实验不过科研人员就没那么好运了,科研中需要用到定量PCR检测的目标广泛,不容易找到带有探针的定量检测试剂盒。当然,针对这种现状,几个大公司陆续推出探针库,比如罗氏的UPL通用探针库,就可以通过组合定量检测出人、大小鼠等7个物种的大部分ORF序列,特别适合大型实验室;ABI公司现在可以根据用户的需要提供约十二万种基因表达引物、探针和两百多万种SNP分析的引物探针进行科学研究,据说是源自于世界各地研究人员要求定制探针或者引物后汇总的结果,可惜就是贵,就连美国的实验室教授
所特有的,下游引物为通用引物就可以了。 荧光定量PCR检测方法有SYBR Green染料法和TaqMan探针法。前者需要调整引物浓度以及引物扩增效率,把引物二聚体调整到越小越好;探针法则需要设计荧光探针,这其中由于MGB探针需要碱基数量少和特异性好的特点而被推荐。 探针设计位置有3个:完全与miRNA序列相同、在miRNA与RT引物的交叉点、完全在RT引物上;这其中又有正向和反向互补两种情况。至于探针要设计在那个位置,根据自己试验情况而定,本人以为效果都差不多。 四、试验操作流程
活组织,分别做尿素酶试验(兰州军医学校生产试剂盒),细菌培养及Warthin-Starry银染检查(由本科专职病理医师完成)。另取胃窦粘膜活组织村本一块,置500μlTE(10mmol/LTris.HCI.1mmol/LEDTA)(pH8.0)缓冲液中研碎,加10%SD30μl蛋白酶K(20mg/ml)3ml,55℃消化1h,以等体积苯酚―氯仿提取模板DNA用于PCR检测,标本处理程中以缓冲液做阴性对照。 统计学处理采用配对四格表资料的X 2 检验
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