Solubilization and preservation of RNA in formamide
Solubilization and preservation of RNA in formamide
Storing RNA in formamide has many advantages over storing it in water or ethanol: (1) formamide will protect the RNA from degradation by RNA enzymes, and RNA stored in this way is quite stable, and can be stored overnight at 4°C or for a considerable period of time at 20°C. The sample can even be left at room temperature overnight without fear of degradation. Samples can even be left at room temperature overnight without fear of degradation; (2) samples can be highly concentrated, with final concentrations of up to 4 mg/ml; and (3) the efficiency of reverse transcription experiments is increased by the availability of highly concentrated RNA samples.
Operation method
Solubilization and preservation of RNA in formamide
Principle
Formamide has active reactivity and special solubility, it can be used as raw material for organic synthesis, paper treatment agent, softener for fiber industry, softener for animal glue, and also used as analytical reagent for determining the amino acid content in rice. In organic synthesis, the majority of uses in medicine, in pesticides, dyes, pigments, spices, additives also have many uses. It is also an excellent organic solvent, mainly used in the spinning of acrylonitrile copolymer and ion exchange resin, as well as anti-static coating or conductive coating of plastic products. It is also used for separating chlorosilanes and purifying oils and fats. Formamide can undergo a variety of reactions, in addition to the three hydrogens involved in the reaction, but also dehydration, de-CO, the introduction of amino groups, the introduction of acyl groups and cyclization and other reactions.
Materials and Instruments
RNA Move I Materials and equipment Caveat 1. When using the CsCl method for RNA extraction, more attention should be paid to the removal of excess salts of RNA. 2. Do not over-dry the RNA in step 4 of the procedure. 3. 1 ul of formamide should be added to the empty internal control when measuring the OD value of RNA to eliminate possible background in the RNA product due to the addition of formamide, and the OD value of the RNA product should be measured using the CsCl method. Common Problems When measuring the OD of RNA, 1ul of formamide should be added to the empty internal control to eliminate possible background in the RNA product due to the addition of formamide. For more product details, please visit Aladdin Scientific website.
Anhydrous ethanol Isopropanol of ethanol Formamide 3mol L of sodium acetate
High speed centrifuge UV spectrophotometer
Distilled and sterilized deionized water is required for all reagent preparations.
1) Anhydrous ethanol
2) Isopropyl alcohol
3) 75% ethanol
4) 100% formamide
5)3mol/L sodium acetate
6) High speed centrifuge
7) Ultraviolet spectrophotometer
II. Methods of operation
1) One-step extraction of RNA using the CsCl method, acid-phenol method, TRIol method or RNaolB method.
2) Precipitate RNA with anhydrous ethanol (CsCl method) or isopropanol (other methods) and centrifuge to obtain RNA precipitate.
3) Wash the RNA precipitate with 75% ethanol to remove excess salts.
4) Air dry, absorb as much ethanol as possible, and make sure there is no excess ethanol present in the RNA.
5) Resuspend the RNA with preserved 100% formamide, the amount of formamide used should depend on the amount of RNA precipitated, usually between 10ul and 1000ul.
6) Most of the RNA will dissolve immediately and can be left at room temperature for 15 min with the pipette tip blowing up and down on the liquid from time to time to promote dissolution of the RNA. If the concentration of RNA is very high, it can be placed at 4℃ overnight to promote complete dissolution.
7) For quantification, dilute 1ul of RNA solution into 500ul of water and measure the OD260 value.
8) If the sample is over-diluted, it can be concentrated by ethanol precipitation as in the case of water-soluble RNA samples, dissolved in water and used.