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说明书：
B0004D-EZ-press RNA purification kit-中文使用指南-2021.pdf
Description
The EZBioscience® EZ-press RNA Purification Kit provides a simple, reliable, and rapid method for isolating high-quality total RNA from a wide variety of sources, without the need for toxic substances such as phenol or chloroform. The kit can be used with cultured cells. Biological samples are first lysed and homogenized in a strong denaturant containing buffer, which immediately inactivates RNases to ensure isolation of intact RNA. After homogenization, ethanol is added to the lysate, creating conditions that promote selective binding of RNA to the silica-based membrane. The sample is then applied to the Spin Column, where the total RNA binds to the membrane, then the membrane is treated with gDNA Remover to eliminate the DNA. Contaminants are efficiently washed away by subsequent step of washing. The high-quality total RNA is then eluted in Elution Buffer. The purified total RNA is suitable for use in a variety of downstream applications, including: RT-PCR, RT-qPCR, and so on.
Components
Components | B0004D (100 Preps) |
Lysis Buffer | 55 ml |
Wash Buffer* | 13 ml |
Elution Buffer | 25 ml |
gDNA Remover | 220 μl |
Spin Columns (with Collection Tubes) | 100 Preps |
*Before using for the first time, add 52 ml 100% ethanol to the Wash Buffer and mix thoroughly.
Storage
Store the gDNA Remover at -20°C. Store other components at room temperature (When using these buffers, be careful to avoid of contamination).
Experimental Procedure at a Glance
Features
1. Fast procedure delivering high-quality total RNA in ~8 minutes.
2. Safe and non-toxic: no phenol/chloroform extraction, or ethanol precipitation.
3. High-quality RNA for downstream applications: RT-PCR, RT-qPCR, and so on.
Representative Experimental Results
Figure 1. RNA isolated from different amounts of A549 cells using EZBioscience® EZ-press RNA Purification Kit and TRIzol. M: 1kb DNA Ladder; Lanes 1 ~ 3: EZ-press RNA Purification Kit; Lanes 4 ~ 6: TRIzol; 1: 2 × 105 cells; 2: 4 × 105 cells; 3: 6 × 105 cells; 4: 2 × 105 cells; 5: 4 × 105 cells; 6: 6 × 105 cells. These results showed that more RNA can be isolated from the same amount of cells by EZBioscience® EZ-press RNA Purification Kit than TRIzol.
Figure 2. Detection of mRNA level of 4 genes (ACTIN, P53, SNAIL and ZO-1) in A549 cells by RT-qPCR (the RNA was purified by EZBioscience® EZ-press RNA Purification Kit and TRIzol method respectively). Figure 2. Shows that, the Ct values of these genes in the RNA sample purified by EZ-press RNA Purification Kit are highly correlated with the RNA purified by TRIzol method (R2=0.9898). And the Ct values from the RNA derived by EZ-press RNA Purification Kit are smaller than by the TRIzol method. These data from Figure 1. and Figure 2. indicate that, EZBioscience® EZ-press RNA Purification Kit can be a good substitution for the TRIzol method in RNA purification. And also, using this Kit can get better results than the TRIzol method.
For detailed information, please check the product manual.
1、建议用6孔板或35mm培养皿培养细胞，细胞密度达到70%以上较好；
2、为了防止吹打细胞时产生过多的气泡，可以在加入裂解液后，在室温放置3~5分钟，使细胞充分裂解，然后吹打10下。
3、为了减少裂解过程中气泡的产生，建议吹打时注意以下细节：吹时液体不要完全吹出，枪头里面可以留一点；吸时，液体也不要全部吸完，防止吸进去气泡。
4、吹打充分以后，可以直接将等体积无水乙醇加入孔板中，吹打10下，将可能出现的沉淀吹散，然后加入离心柱中，4000g（大约相当于eppendorf或Thermo离心机的7000rpm）离心1min，弃去液体；
5、DNA酶可以用ddH2O稀释，或者用elution buffer稀释，然后加入柱中央的膜上，室温放置5分钟，然后不要离心，直接加入wash buffer后，12000g离心1分钟；
5、洗涤后，建议空柱离心一次，以充分去除可能残留的wash buffer；
6、洗脱时，洗脱液一定要加到柱子中央的膜上，不能加到侧壁上，否则会因为RNA没有被溶解导致产量大大减少；
7、溶解时建议先放2分钟，离心1分钟，然后将液体吸出来，加入离心柱中央，再放置3分钟，使RNA充分溶解，然后离心。离心后，RNA应迅速转移到冰上放置并测浓度，进行后续实验。
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