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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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
这里所说的一个碱基的差异该怎么检测?如何理解?
原理?(没有就算了...)
答的好的提高悬赏50分
有原理的追加50分
谢谢啊~~~~
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
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