|Supply Ability:||100 Piece/Pieces per Month|
|Place of Origin:||Jiangsu China|
|Storage:||15 ℃ - 25 ℃|
|name:||Total RNA Kit / animal tissue extraction kit|
|Model Number:||Total RNA Kit / animal tissue extraction kit|
|Packaging Detail:||Plastic bottle: 50T / bottle Need other packing, please contact customer service|
Total RNA Kit / animal tissue extraction kit
|Place of Origin||Nanjing, China (Mainland)|
|Supply Capacity||100 Piece/per Month|
|Other packaging||please consult customer service|
The improved guanidine isothiocyanate / phenol one-step method can cleave cells and inactivate RNase. Then total RNA is selectively adsorbed on the silicon matrix membrane in the centrifugal column in the state of high dissociation salt. Then, through a series of rapid rinsing centrifugation steps, the eluent will remove cell metabolites, proteins and other impurities. Finally, the pure RNA will be eluted from the silicon matrix membrane by low salt RNase free water.
1. All the silicon matrix membranes in the centrifugal adsorption column are made of imported special adsorption membranes, and the difference of adsorption capacity between columns is very small, and the repeatability is good. It overcomes the disadvantage of unstable membrane quality of domestic kits.
2. Combined with the advantages of guanidine isothiocyanate / phenol one-step reagent stability, high purity and convenient centrifugal column, RNA can be directly eluted from the centrifugal column without isopropanol precipitation and ethanol washing process, avoiding the problem of over drying and insoluble.
3. The unique R lysate formula can effectively eliminate genome pollution.
4. The purity of RNA was higher by washing the deproteinization process for many times.
5. The content of 5S in total RNA was effectively removed and the purity was improved.
matters needing attention
1. Before using for the first time, please add the specified amount of ethanol into the RW bottle of rinsing solution. After adding, please mark the added ethanol in time to avoid adding it for many times!
2. In order to prevent RNA degradation, all centrifugation steps were carried out at 4 ℃ unless otherwise specified. Use traditional desktop centrifuges with speeds up to 13000 rpm, such as Eppendorf 5415c or similar.
3. There are irritant and harmful compounds in lysate R. latex gloves should be worn during operation to avoid contamination of skin, eyes and clothes. If it is contaminated with skin and eyes, wash with plenty of water or normal saline.
4. Considering the environmental protection, the kit does not contain chloroform, which is commonly used in the laboratory. Users need to prepare chloroform before use.
5. the conventional agarose gel electrophoresis and denaturing gel electrophoresis can be used to analyze the quality of RNA. After electrophoresis, two dominant ribosomal RNA bands, i.e. ~ 5KB (28s) and ~ 2KB (18S), can be seen in a good RNA product. The brightness ratio of the bands is about 2:1. Sometimes we can see ~ 0.1kb and 0.3kb (5S, tRNA) bands. But sometimes it is normal to see 4 or 5 bands according to different species such as some plant tissues. If immature RNA precursor or heterogeneous nuclear RNA or small nuclear RNA are extracted, discontinuous high molecular weight bands between 7KB and 15KB may also be seen.
6. When detecting the absorbance ratio of od260 / od280, RNA sample should be dissolved in te for detection. If the sample is diluted with water, the od280 will increase and the ratio will decrease due to the low ionic strength and pH value of water.
7. After adding lysate R, the sample can be stored at – 60 ℃ - 70 ℃ for more than one month before adding chloroform.
Storage and stability
1. All solutions should be clear. If the ambient temperature is low, the solution may form precipitation, and it should not be used directly. It can be heated in a 37 ℃ water bath for a few minutes to restore clarification.
2. Improper storage at low temperature (4 ℃ or - 20 ℃) will cause solution precipitation and affect the use effect. Therefore, transportation and storage are carried out at room temperature (15 ℃ - 25 ℃). The pyrolysis liquid R can be transported at normal temperature and stored away from light at 4 ℃.
3. Avoid volatilization, oxidation and pH value change of reagents exposed to the air for a long time, and close the lid of each solution after use.
Tip: Please add the specified amount of ethanol into the RW bottle of rinse solution before the first use!
a. Tissue: mix the tissue samples with glass or strong homogenizer, and store them in liquid nitrogen. The samples need to be ground in a mortar. Add 1 ml of cell lysate r to each 50-100 mg tissue for homogenization. The volume of tissue sample should not exceed 10% of the volume of cell lysate R.
b. Monolayer growth cells: directly add 1 ml cell lysate r into the culture plate with a diameter of 3.5 cm to dissolve the cells, and gently blow and mix with a pipette gun. The amount of cell lysate R (1ml per 10cm2) was determined according to the area of the culture plate rather than the number of cells. In general, 1 ml cell lysate R is added into an ordinary cell culture bottle, which is shaken rapidly to make the lysate r fully contact with all cells at the bottom of the bottle, inactivate RNase, blow repeatedly with a pipette gun and mix well. When the amount of R in cell lysate is insufficient, the extracted RNA will be contaminated with DNA.
c. Suspended growing cells: cells are precipitated by centrifugation. In the cell lysate r reagent, the cells were broken by repeated blowing with a pipette gun. 1 ml cell lysate r was added to every 5-10 × 106 animal cells, plant or yeast cells or 1 × 107 bacteria. Washing cells should be avoided before adding lysate R, otherwise the possibility of mRNA degradation will be increased. The use of a homogenizer may be required to break down certain yeasts and bacteria.
2. The homogenate was shaken violently and then incubated at 15-30 ° C for 5 minutes to completely decompose the ribosomes.
3. Optional steps: centrifugation at 12000 rpm at 4 ° C for 10 minutes. Carefully remove the supernatant and transfer it into a new RNase free centrifuge tube. When the sample is rich in protein, fat, polysaccharide or extracellular material such as muscle. An additional separation step may be required for adipose tissue or tuber parts of plants. After homogenization, centrifugation at 12000 rpm for 10 minutes at 2 ~ 8 ° C was performed to remove the insoluble substances from the homogenate. The remaining precipitates contained extracellular membrane, polysaccharide and high molecular weight DNA, while the upper layer of the superplankton contained RNA.
4. Add 0.2 ml chloroform to 1 ml cell lysate R. Cover the sample tube tightly, shake vigorously for 15 seconds, and place it at room temperature
Nanjing Duly Biotech Co.,Ltd
has been established for 11 years. It is mainly engaded in biological and chemical reagents,including enzyme reagents, proteins, antibiotics, plant hormones, nucleic acids, pigments, amino acids, vitamins,carbohydrates, buffers, surfactants,culture media and Separation of materials and kits.There are more than 10,000 products in total.
Our company’s aim is to provide customers with the best quality products and the most intimate service.Personnel has been trained; every batch of material is checked; each procedure guarantees the quality; every bag of our products is top grade;service is thoughtful all the time.The quality, colors and forms of our products have reached a high level in related chemical industry, which relies on characteristic and advanced processing technology and it has gained a high and reliable reputation from customers at both quality and services.