EZ spin Plus Tissue/cell Fast RNA kit
2024-11-29EASYspin Fast Blood RNA Kit
2024-11-29EASYspin RNA Mini Kit
Product Number: RNK0701
Shipping and Storage
1.All solutions should be clear. If the ambient temperature is low, the solution may form precipitates and should not be used directly. It can be heated in a 37℃ water bath for a few minutes to restore clarity.
2.Inappropriate storage at low temperatures (4℃ or -20℃) can cause solution precipitation, affecting the effectiveness of use. Therefore, transportation and storage are carried out at room temperature (15℃ -25℃).
3.To avoid volatilization, oxidation, and pH changes caused by prolonged exposure of reagents to the air, each solution should be covered tightly in a timely manner after use.
Components
| Component | Storage | RNK0701 50 Preps |
| Buffer RLT | RT | 50ml |
| Buffer RW1 | RT | 40 ml |
| Buffer RW | RT | 10ml |
| RNase-free H2O | RT | 10 ml |
| 70% ethanol | RT | 9ml RNase-free H2O |
| RNase free adsorption column RA and collection tube | RT | 50 |
Description
The unique Buffer RLT rapidly cleaves cells and inactivates cellular RNA enzymes. After adjusting the binding conditions with ethanol, RNA selectively adsorbs onto the silica matrix membrane in a highly dissociated salt state. Through a series of rapid rinsing centrifugation steps, Buffer RW1 and Buffer RW remove impurities such as cellular metabolites and proteins. Finally, low salt RNase free H2O elutes pure RNA from the silica matrix membrane.
Features
1.Completely do not use toxic reagents such as phenol, chloroform, Beta mercaptoethanol, and do not require steps such as ethanol precipitation.
2.Fast and simple, the operation of a single cell sample can generally be completed within 10 minutes.
3.Multiple column washes ensure high purity, with a typical OD260/OD280 ratio of 2.1-2.2 (the ratio of 100% pure RNA is generally around 2.2. Many companies' products have reduced ratios due to residual proteins or DNA, which cannot meet the purity standard of 2.2. Therefore, reducing the requirement by 1.9-2.0 is sufficient for use, but our product standards can generally reach high levels of 2.1-2.2 purity).
Note
1.All centrifugation steps are completed at room temperature, using a desktop centrifuge with a speed of up to 13000 rpm is sufficient.
2.The sample processing capacity should never exceed the RNA adsorption column RA processing capacity, otherwise it may actually lead to a decrease in yield. There is a significant difference in RNA content among different types of tissue cells, and COS cells have abundant RNA content. Cells exceeding 3×106 will exceed the column adsorption capacity. So when starting to explore the experimental conditions, if the RNA content of the sample is not clear, it is better to use a smaller sample processing volume, such as cells not exceeding 3-4×106 and tissues not exceeding 10mg. In the future, the processing capacity will be increased or decreased based on the sample testing situation.
3.Buffer RLT and Buffer RW1 contain guanidine hydrochloride/guanidine isothiocyanate compounds. When operating, latex gloves should be worn to avoid contact with skin, eyes, and clothing. If it gets on the skin or eyes, rinse with plenty of water or physiological saline.
4.To prevent RNase pollution, attention should be paid to the following aspects:
4.1.Regularly replace with new gloves. Because the skin often carries bacteria, it may lead to RNase contamination.
4.2.Use RNase free plastic products and gun heads to avoid cross contamination.
4.3.RNA is not degraded by RNase in Buffer RLT. However, plastic and glassware without RNase should be used during the extraction and subsequent processing. Glassware can be baked at 150℃ for 4 hours, while plastic containers can be soaked in 0.5M NaOH for 10 minutes, then thoroughly washed with water and sterilized to remove RNase.
4.4.Prepare the solution using water without RNase. (Add water to a clean glass bottle, add DEPC to the final concentration of 0.1% (v/v), leave at 37℃ overnight, and sterilize under high pressure.)
5.Regarding trace residues of DNA:
Generally speaking, any total RNA extraction reagent cannot completely avoid trace residues of DNA during the extraction process (DNase digestion cannot achieve 100% residue free). Our EASYspin RNA Mini Kit extraction product, due to the use of our unique buffer system and genomic DNA clearance column technology, the vast majority of DNA has been cleared and does not require DNase digestion. It can be directly used for reverse transcription PCR and fluorescence quantitative PCR. In some special cases, such as residual DNA caused by excessive DNA content or strict mRNA expression analysis requiring fluorescence quantitative PCR, we suggest that when selecting templates and primers:
5.1.Select primers that cross introns to cross the connections in mRNA, so that DNA cannot serve as a template for amplification reactions.
5.2.Select primer pairs with different sizes of amplified products on genomic DNA and cDNA.
5.3.Treat RNA extract with DNase I of RNase free to improve efficacy. This reagent kit can also be used for RNA cleaning up after DNase I treatment. Please contact us for specific operating instructions.
5.4.Before rinsing with Buffer RW1, perform DNase I treatment directly on the adsorption column RA. Please contact us to request specific operating instructions (DNase Digestion Kit:RNK3401).
6.RNA purity and concentration detection:
6.1.Integrity: RNA can be detected by ordinary agarose gel electrophoresis (electrophoresis conditions: gel concentration 1.2%; 0.5×TBE electrophoresis buffer; 150v, 15min). Due to 70% -80% of RNA in cells being rRNA, very clear rRNA bands should be visible under UV after electrophoresis. Animal rRNAs are approximately 2 kb and 1 kb in size, equivalent to 28 S and 18 S rRNAs, respectively. The maximum rRNA brightness in animal RNA samples should be 1.5-2.0 times the brightness of the second largest rRNA, otherwise it indicates degradation of the RNA sample. The appearance of small dispersed flakes or disappearance of bands indicates severe degradation of the sample. But attention should be paid to distinguishing whether the extracted RNA sample itself degraded or extracted.
6.2.Purity: The OD260/OD280 ratio is a reference indicator for measuring the degree of protein contamination. High quality RNA, OD260/OD280 reading between 2.1 and 2.2. The ratio of 100% pure RNA is generally around 2.2 (the ratio of 100% pure RNA is usually around 2.2). Many companies' products have a lower ratio due to residual proteins or DNA, which cannot meet the purity standard of 2.2. Therefore, reducing the requirement by 1.9-2.0 is sufficient for use, but our product standards can generally reach a high level of 2.1 to 2.2 purity). The OD260/OD280 reading is influenced by the machine used for measurement and also by the pH value of the dilution solution used for measurement. Microspectrophotometers generally do not require dilution and are not affected by the pH value of the diluted solution. However, for the same RNA sample, if the machine requires dilution during measurement, assuming that the OD260/OD280 reading measured in a 10mM Tris, pH 7.5 dilution solution is between 1.9-2.1, the reading measured in an aqueous solution may be between 1.5-1.9, but this does not mean that the RNA is impure.
6.3.Concentration: Take a certain amount of RNA extract, dilute it n times with RNase free water, adjust the spectrophotometer to zero with RNase free water, take the diluent for OD260 and OD280 measurements, and calculate the RNA concentration according to the following formula: final concentration (ng/μl)=(OD260)×(dilution n)×40.
