BP-10 Spin Column Genomic DNAMinipreps Kit, Plant
BP-10 Spin GenomicDNA Minipreps Kit, Plant | GDMIP-100P, 100 Preps |
RNase A (10 mg/ml)(a) PCLSolution PPSolution PBSolution WashSolution(b) ElutionBuffer(c) EZ-10 SpinColumn & 2.0-ml Collection Tube Protocol | 300 ml 30 ml 4 ml 40 ml 24 ml 10 ml 100 1 |
Introduction
The BP-10Spin Column Kits provide a simple and efficient method for purification ofgenomic DNA from different sources such as Bacteria, Plant, Animal and Blood.
The DNA isselectively adsorbed in silica gel-based BP-10 Spin Column and other componentsare washed away. The genomic DNA is eluted off the column and can be used forany downstream applications, including restriction enzyme digestion, PCR,Southern-blotting, etc.
Thepurification methods used in these protocols do not require use of phenol,chloroform, or CsCl. The DNA is purified without an additional step of ethanolprecipitation.
These kitsare designed for research only. The purified DNA should not be used for liveanimal transfections. It is also not to be used for human diagnostic or drugproduction purposes.
Features
- Simple, fast and efficient
- Preparation of high quality genomicDNA from various sources
- High yield and reproducible
- No phenol / chloroform extraction orethanol precipitation required
- High capacity: up to 10µg of DNA percolumn
Applications
Efficient ofup to 10µg of genomic DNA purification from different sources
Storage
With theexception of the Proteinase K, the kit may be stored at room temperature.Proteinase K should be stored at 4ºC for short term or -20ºC for longterm. The kit is stable for 12 months at room temperature. For longer storage,keep all contents in cold place.
Quality Control
Each lot ofBP-10 Spin Column kit is tested against predetermined specifications to ensureconsistent product quality.
- PCLSolution DOES NOT contain RNase A (100 µg/ml). Please add entire contents ofRNAse A into PCL Solution and store at 4ºC for long term storage. PCL Solution may form a precipitate upon storage. If necessary,dissolve the precipitate by warming up to room temperature.
- Beforeuse, add 96ml of 100% ethanol to 24ml Wash Solution for GDMIP-100P.For other volumes of wash solution, simplyadd enough ethanol to make a 4:1 ratio (volume of added ethanol : volume of WashSolution = 4:1).
- ElutionBuffer is 2.0mM Tris-HCl pH 8.0~8.5. Although TE buffer pH 8.0 or water can beused, yield may be 20% lower.
Procedures for Isolation of Genomic DNA from Plants
- PlantTissue Sample Preparation
- Grind plant tissue under liquid nitrogen to a finepowder using a mortar and pestle. Transfer the powder and liquid nitrogen to1.5ml Eppendorf tube and allow liquid nitrogen to be evaporated. Do not allowthe sample to thaw. Proceed immediately to Step 2.
- Note:
- Ifsample can not be used immediately for genomic DNA extraction, it isrecommended to store at -20ºC for long-term use.
- Avoid repeated freezing and thawing of stored samples, since this leads to reduced DNA size and yield.
- Incubation period depends on the nature of sample. Longer period incubation even overnight incubation will not affect the result.
- Better results could be achieved if starting material is less than 60mg.In any case, the quantity of samples should not exceed 100 mg for one BP-10 Spin Column.
- Checkthe volume of grounded material, and add equal volume (approximately 150µl) ofPCL Solution (Plant Cell Lysis Solution).
- Vortexand shake the tube several times. Incubate at 65ºC for 20 minutes,vortex or pipette up and down to further remove any clumps. Clumped tissue willnot lyse properly and will result in a lower yield of DNA.
- Add25µl PP Solution. Mix well. Keep the solution on ice for 15 minutes.
- Centrifugeat 4ºC, 8,000 x g (10,000 rpm) for 5 minutes. Apply the clearlysate to an BP-10 Spin Column.
- Add300µl PB buffer to the BP-10 Spin Column. Mix gently by inverting the tube.Incubate the mixture for 3 minutes at room temperature. During incubation, mixoccasionally by inverting the tube.
- Centrifugeat 4ºC, 8,000 x g(10,000 rpm) for 2 minutes.
- Discardthe flow-through in the Collection Tube. Add 500µl of Wash Solution, and spinat 8,000 x g (10,000 rpm) for 2 minutes.
- RepeatStep 8.
- Discard flow-through. Spin at 8,000 xg (10,000 rpm) for an additional minute to remove residual amount of WashSolution.
- Place the column into a clean 1.5mlEppendorf tube. Add 30-50µl Elution Buffer into the center part of membrane inthe column. Incubate at RT for 2 or 3 minutes. Incubating the tube at 37 or 50ºC for 2 minutes may increase recovery yield.
- Spin at 8,000 x g (10,000 rpm) for 2minutes to elute DNA from the column. Measure DNA quantity by UV absorption atA260 (1.0 OD unit is equivalent of 50µg).
- For long term storage, keep aliquotsof purified genomic DNA at -20ºC.
Measure DNA quantity by UV absorption at A260(1.0 OD unit is equivalent of 50 µg). Assess genomic DNA quality by ananalytical 0.7% agarose gel. Isolated genomic DNA should not contain RNA. Itslength should be over 50 kb.
Troubleshooting Guide: BP-10 SpinColumn Genomic DNAMinipreps Kit, Plant
- Low Yield
- There are a number of variables that can cause low yield.
- Inefficienthomogenization.
- Ensure that the material is completely disrupted. If necessary, increase time of homogenization.
- Low DNA content of plant tissue.
- Increase the amount of starting material to 200mg.
- RNA contamination
- RNase activity is weakened or lost.Add 30% additional RNAse A, and store solution at 4ºC.
- OD 260nm/280nm ratio outside1.9-2.2 range
- If the ratio of OD260nm/280nm is greater than 2.2, there maybe traces of ethanol present. If the ratio of OD260nm/280nm is smaller than1.9, there is a chance of protein contamination. Make sure the sample is mixedwell after Proteinase K digestion.
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同一温度下，首先通过M-MLV反转录酶产生靶标核酸（RNA）的一个双链DNA拷贝，然后利用T7RNA多聚酶从该DNA拷贝上产生多个（100～1000个）RNA拷贝；每一个RNA拷贝再从反转录开始进入下一个扩增循环；还可以加入荧光探针（分子信标），带有荧光标记的探针和这些RNA拷贝特异结合，产生荧光。该荧光信号可由实时荧光PCR仪实时捕获，直观反映扩增循环情况。
SAT检测技术的优势
→先进的恒温扩增技术
核酸扩增在一个温度下进行（42℃），无需热循环。
→领先的RNA检测技术
SAT技术直接以病原体特异性RNA为扩增靶标，以扩增产物RNA为检测靶标，在实际应用中更显优势意义。
→更高的检测灵敏度和准确性
SAT技术的扩增效率极高，15～30分钟即可将模板扩增109倍，检测灵敏度和准确性远高于其他核酸检测技术。扩增时间短，效率高。
→有效减少假阴性结果
SAT技术采用M-MLV逆转录酶和T7RNA多聚酶进行核酸扩增，相对于其它核酸扩增技术，反应抑制物更少，有效减少假阴性结果。
→有效的解决了扩增产物的污染问题
由于扩增产物为RNA，环境中极易降解，从而大幅度提高检测结果的可靠性。
→大大降低了核酸扩增实验室的要求
1．目的
学会PCR操作的基本技术。
2．原理
是将待扩增的DNA模板加热变性，与其两侧互补的寡聚核苷酸引物复性，然后经过耐热的DNA聚合酶延伸。再进入下一轮变性—复性—延伸的循环，n次循环后DNA可被扩增（1+X）n倍。其中<25nt的引物退火温度Tm=2（A+T）+4（G+C）。
3．器材
旋涡混合器，微量移液取样器，移液器吸头，0.2mlPCR微量管，双面微量离心管架，PCR仪，台式离心机，琼脂糖凝胶电泳系统，水漂，恒温水浴。
4．试剂
5U/μlTaqDNA聚合酶，PCR缓冲液（10×，MgCl2free），25mMMgCl2，dNTP，引物，模板质粒pBS-CHI，无菌ddH2O。
5．实验准备
dNTP混合液（每种25mM），TAE电泳缓冲液，荧光染料，10´加样缓冲液，1.5ml离心管装入铝制饭盒（灭菌）、移液器吸头装入相应的吸头盒（灭菌）。合成的引物：Senseprimer5＇-GGATCCACAATGATGAGAGCC-３＇
Antisenseprimer５＇-GATATCATGGTGAGGTAGCTAGCTT-３＇
目的基因模板质粒：已经克隆在pBS载体上的1128bp几丁质酶（chitinase，CHI）基因。
待扩增的CHI片段长度：996bp。
6．操作步骤
（1）在0.2mlPCR微量离心管中配制50μl反应体系。（以下加样量供参考，括号内是最终需要量，实验时需参照Taq酶说明书计算）
ddH2O32μl
10×PCRbuffer（不含MgCl2）5μl
25mMMgCl23μl
2.5mmol/LdNTP4μl（每种dNTP终浓度0.2mM）
10μmol/LPrimer12μl（12.5~25pmoles）
10μmol/Lprimer22μl（12.5~25pmoles）
模板质粒1μl（1×10-3pmoles）
5U/μlTaq酶1μl（5U）
总体积50μl
（2）根据厂商的操作手册设置PCR仪的循环程序：
①94℃5min
②94℃1min
③54℃1min
④72℃1min50s
⑤goto②29times
⑥72℃10min
（3）PCR结束后，取10μl产物进行琼脂糖凝胶电泳。观察胶上是否有预计的主要产物带。
（4）清理桌面，撰写实验报告。
（5）PCR产物可以直接与T载体连接（见实验五），然后转化感受态细胞（见实验八）。
7．思考
（1）复性温度如何确定？
（2）为什么要在最后延伸10min？
（3）是否有非特异性扩增产物（或引物二聚体），如何才能消除？
更多资讯请关注于http://www.chinazglab.com/
我做的是用锁式探针来检测dna模板的ram，探针80base，其中杂交区40base，引物分别为16、18base，连接酶选取的T4 ligase和Taq DNA ligase两种，分别对应恒温和变温情况下的扩增。水解体系为EXO I和Exo Ⅲ的混合体系，参考的外文文献，应该没什么问题。聚合酶用的是Bst DNA大片段聚合酶。
一开始选择的探针浓度为200pmol/L，扩增效果挺好，当时用的DNA模板是质粒阳性标准品，但是后来在做敏感性的时候发现条带梯度差异几乎没有，而且阴性也出带，后来就没法做敏感性和特异性验证了，实验停滞了2个月一直也没进展。
按理说RAM借助锁式探针扩增的独特方式以及水解体系的存在可以有效防止污染才对，希望大家给点建议该如何解决。
万分感谢！！！！！！！！！！
附一张以前做敏感性的图（marker为2000的DNA ladder）以做参考
今天发现，我在核酸技术区开辟的“DIG/Biotin标记的Southern/Northern杂交专题讨论”4个月，今天回帖数破100，点击达1500多次。能和大家一起分享和交流，并且得到共同提高，心情很是愉快。
所以想借PCR技术区的宝地，新开辟“环介导等温扩增技术(Loop-MediatedIsothermalAmplification,LAMP)专题讨论”。我做LAMP一年多了，有些经验。而最近，和我谈起LAMP的朋友明显增多。所以在这方面大家有什么心得、体会、资料和实验困难，希望能一起交流讨论。谢谢支持！
(2)操作多份样品时，应先配制反应混合液并分装，减少操作，避免污染，同时增加反应的精确度。试剂的配置和分装应在装有紫外灯的超净工作台或负压工作台操作。
(3)移液器和吸头需专用。由于移液器极易受气溶胶或模板核酸的污染，移液器应尽可能使用可替换或可高压处理的，吸头尽可能使用带滤芯的吸头。
(4)防止操作人员污染，使用一次性手套，EP管与吸头应一次性使用，吸头不要长时间暴露于空气中，避免气溶胶的污染。
(5)加样或吸取模板核酸时要十分小心，吸样要缓慢，防止样品进入移液器内；吸样时尽量一次性完成，忌多次抽吸，以免交叉污染或产生气溶胶污染。
(6)EP管打开应先离心，将管壁及管盖上的液体甩至管底部。开管动作要轻，以防管内液体溅出。若不小心溅到手套或桌面上，应立刻更换手套并用稀酸擦拭桌面。
(7)模板核酸应密封保存，防止外溢及外来核酸的进入。
(8)设立适当的阴阳性对照及重复实验，既可验证LAMP反应的准确性，又可以协助判断扩增系统的可信性。
(9)扩增产物应妥善处理，尽量避免开盖检测，开盖极易产生气溶胶污染。本公司的试剂均采用闭管检测，有效防止污染发生。展开
