Factors that affect the activity of restrictive endoenzymes.
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Last Update: 2020-10-29
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Source: Internet
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Author: User
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. DNAPurity in DNAsamples contains
protein
, or does not remove ethanol, EDTA , SDS, phenols, chloroform, and certain high concentrations of metal ions used in clean preparation, which can reduce the catalytic activity of the limit enzyme and even prevent the restriction enzyme from working.. 2buffer
nucleic acid intrinsic limitase of nucleic acid infusion limitase standard buffer includes magnesium chloride, sodium chloride or potassium chloride, Tris-HCL , -based ethanol or(
DTT
)and bovine
serum
albumin(BSA) , etc.. Use a buffer that limits the activity of all restriction enzymes.. Different
limiting endoenzymes
have different naClconcentrations, which is a major difference in the composition of different limiting enzyme buffers. According to this can be divided into high-salt, medium-salt and low-salt buffer, in the double-enzyme or multi-enzyme solution, if these enzymes can be cut in the same buffer good, then several enzymes can be enzymatic at the same time, if these enzymes require a different buffer, can be digested in three ways: first with the requirements of low-salt buffer restrictive endolytic enzyme digestion DNA , then replenish the appropriate amount of NaCl , then digest it with a limit enzyme that requires a high-salt buffer;is first enzymaticized with one enzyme, then the product is dissociated with ethanol precipitation enzymes, and then resurred in another buffer for a second enzymatic solution.. 3the temperature of the enzyme cutting digestion reaction
the temperaturethe DNAthe digestive reaction is an important factor affecting the activity of the endoenzyme. Different nucleic acid in-cut limit enzymes have their own most appropriate reaction temperature. The most appropriate reaction temperature for most restricted endoenzymes is 37 degrees C , and the most appropriate reaction temperature for a few restricted endoenzymes is above or below 37 degrees C .. The molecular structure DNAmolecular configuration of DNAhas a significant effect on the activity of the nucleic acid intracion limiting enzyme, such as digestion of ultra-helix DNA is many times higher than the amount of digestion linear DNA . Some restriction enzymes also have significant differences in efficiency in digesting their own restricted sites in different locations.. The termination of the restrictive endoenzyme reactionusually 5min5minunder65 ;the termination reaction fluid (e.g. EDTAof 0.5mol/L ) to the final concentration in the solution to 10 mol/L ) to chelate Mg2
to terminate the reaction.
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