Deoxyribonuclease cleaves RNA at a specific site
Deoxyribonucleases are oligodeoxyribonucleotides consisting of about three dozen deoxyribonucleotides, which are easy to prepare and convenient to use, and are an effective method for in vitro cleavage of RNA at specific sites. Deoxyribonucleases commonly used for in vitro cleavage of RNA substrates are "10~23" type deoxyribonucleases.
Operation method
Deoxyribonuclease cleaves RNA at a specific site
Materials and Instruments
Deoxyribonuclease DMA enzyme without RNAase Cutting substrate RNA Move I. Materials and equipment Caveat 1) The low salt concentration of the reaction system favors the specific binding of deoxyribonuclease to the substrate RNA. It also reduces non-specific binding.2) If the optimal reaction conditions for the cleavage reaction of different substrate RNAs and small deoxyribonucleases are not exactly the same, the K-adapted reaction conditions can be obtained by a small reaction volume test, and then large-scale preparation can be carried out. Reaction conditions that need to be optimized include: magnesium ion concentration, ratio of substrate RNA to deoxyribonuclease, concentration of deoxyribonuclease, reaction temperature, and reaction time.3) Urea can be added to the reaction buffer for some specific reactions.4) According to the different binding sequences of DNAzyme and substrate RNA, deoxyribonucleases are divided into two categories: fast reaction and slow reaction. For fast-reacting enzymes, the substrate RNA can be completely cleaved by reacting at 37°C for 5 min; for slow-reacting enzymes, the cutting efficiency can only reach about 40% by reacting at 37°C for 4 h. The reaction time can be adjusted by adding urea in the reaction buffer. For more product details, please visit Aladdin Scientific website.
5X reaction buffer 5X magnesium acetate or magnesium chloride Anhydrous ethanol Water-saturated phenol
1) Deoxyribonuclease (see section on deoxyribonucleases for design and format)
2) 5X reaction buffer: 750Tnmol/LNaCl, 200 mmol/LTris-HCl (pH 8.0)
3)5X Magnesium acetate or magnesium chloride: 300 mmol/L
4) DMA enzyme without RNAase
5) Cutting substrate RNA
6) Anhydrous ethanol
7) Water-saturated phenol
II. Methods of operation
1) Deoxyribonuclease cleavage of substrate RNA reaction system: add substrate RNA (final concentration of 1~10umol/L, deoxyribonuclease (final concentration of 30umol/L), reaction buffer (final concentration of 15 mmol/LNaCl), 4 mmol/LTris-HCl (pH8.0), and a buffer of 4 mmol/LTris-HCl (pH8.0), and a buffer of 4 mmol/LTris-HCl (pH8.0), and a buffer of 4 mmol/LTris-HCl (pH8.0).
2) Denature the RNA substrate and deoxyribozyme by heating at 95°C for 3~4 min, then place on ice for 5 min.
3) incubate at 25℃ for 10 min.
4) Add 5X reaction buffer to bring the concentration of reaction buffer to that of 1X reaction buffer. Increase the reaction temperature to 37~42℃.
5) Add 5X magnesium acetate or magnesium chloride to make the concentration of magnesium ion reach 60 mmol/L, so as to start the cleavage reaction of deoxyribonuclease.
6) Depending on the substrate RNA and deoxyribonuclease, the reaction time was 30 min~4 h. The reaction temperature was raised to 37~42℃.
7) At the end of the reaction, place the reaction tube on ice and recover the RNA by ethanol precipitation.
8) If the deoxyribonuclease interferes with the subsequent reaction, after ethanol precipitation, dissolve 100ul of RNA-free water, add 15ul of DNA enzyme, react at 37℃ for 1h, extract with phenol, precipitate with ethanol, and then isolate and purify by denaturing polyacrylamide gel.