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低温
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- 供应商:
上海圻明生物
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50次
Piscirickettsia salmonis鲑鱼立克次氏体探针法荧光定量PCR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-150bp的产物长度。3. 标准曲线线性关系不佳 加样存在误差: 使得标准品不呈梯度。 标准品出现降解: 应避免标准品反复冻融,或重新制备并稀释标准品。 引物或探针不佳: 重新设计更好的引物和探针。 模板中存在抑制物,或模板浓度过高4. 负对照有信号 引物设计不够优化:应避免引物二聚体和发夹结构的出现。 引物浓度不佳:适当降低引物的浓度,并注意上下游引物的浓度配比。 镁离子浓度过高:适当降低镁离子浓度,或选择更合适的mix试剂盒。 模板有基因组的污染:RNA提取过程中避免基因
200μl (2)冰浴30min至1h。中间摇动3次,以防受体菌沉底。 (3)42℃90s。 (4)37℃水浴5min。 (5)加入无相应抗生素的Lb 200μl,混匀后,37℃水浴30~60min。 (6)分别取3组反应物各50μl在含相应抗生素的固体LB培养基平皿上涂布,室温下干燥,然后倒置于37℃温箱中培养过夜。 (三)转化子的快速鉴定-快速细胞破碎法 (1)挑取转化平板上的单菌接种于含相应抗生素的2ml LB中,37℃振荡培养至A590 值为0.6~0.8。 (2)取1ml
受体菌对照组 200μl 0 200μl 转化组 190μl 10μl 200μl (2)冰浴30min至1h。中间摇动3次,以防受体菌沉底。 (3)42℃90s。 (4)37℃水浴5min。 (5)加入无相应抗生素的Lb 200μl,混匀后,37℃
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