ã€Fundamental】 There are more than one hundred restriction enzymes, each of which has its specific nucleotide sequence recognition specificity, and the activity of the enzyme needs to be activated by Mg+2. There are also many differences between different enzymes: some enzymes require activation of other cofactors such as ATP in addition to Mg+2; the distance between the cleavage site and the recognition sequence is different; some endonucleases also have methylation. Based on these differences, restriction enzymes can be classified into types I, II, and III. Type II restriction enzymes only require the activation of divalent magnesium ions, which cleave double-stranded DNA within their recognition sequences, produce various DNA fragments with the same terminal structure, and most type II enzymes provide viscosity The end facilitates fragment rejoining, and the sequence recognized by most type II enzymes has an inversely symmetric structure, or a palindrome. Identifications and incisions such as EcoRI and HindIII are: EcoRI : G ↓AATT C HindIII : A↓AGCT T T TCGA↑ AC TTAA↑G The number of cleavage fragments produced by the restriction endonuclease on the circular plasmid DNA is consistent with the number of nicks. Therefore, by identifying the number of bands in the electrophoresis gel after fragmentation, the number of incisions can be inferred; the fragment size can be determined from the fragment mobility. Using the linear DNA of known molecular weight as a control, the relative molecular size of the unknown DNA having the same molecular shape can be roughly measured by comparison of electrophoretic mobility. The relative molecular weight (Mr) of plasmid DNA is generally in the range of 106-107. For example, the relative molecular mass of plasmid pBR322 is 2.8×106, and there are three configurations in the cell: 1 covalent closed-loop DNA, often in the form of supercoil; 2 If one of the two strands breaks in one or more strands, the molecule can rotate to eliminate the tension of the strand. This relaxed molecule is called open-loop DNA; 3 double-stranded linear DNA is cut by two strands. It is cut at the same site, can not be looped, and is completely open to a linear shape, referred to as linear DNA. If the relative molecular weight of the plasmid DNA is to be determined, it is preferred to hydrolyze the plasmid with a single nick to obtain a linear DNA fragment. The migration speed of plasmid DNA of three configurations during electrophoresis was: covalent closed-loop DNA>linear DNA>open-loop DNA. In this experiment, the restriction enzyme BamHI was used to cleave the 1200 bp KanR gene fragment inserted into the pUC18-KanR plasmid. ã€equipment】 Water bath 2. Autoclave ã€Reagents】 1.10×restriction enzyme buffer 2. Restriction enzyme BamH I 3. DNA sample, recombinant plasmid (see above) ã€Steps】 1. Add the following reagents to the Ep tube: 10× restriction enzyme buffer 2μl pUC18-KanR 2μl ddH2O 15μl BamH I 1μl 2. Mix the upper liquid and incubate at 37 ° C for 1 h. 3. The reaction was stopped by adding 1 μl of 0.5 mol/L EDTA (pH 8.0). 4.1% agarose gel electrophoresis, detection of enzyme digestion results. Shanghai Chuangsai Technology has excellent performance, interleukin cytokines, fetal bovine serum, electrophoresis equipment scientific instruments, raw material drug standards, chemical reagents, cell culture consumables, Shanghai Chuangsai, mass products special promotions, welcome to inquire! Memo Pad
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The basic principle and operation steps of restriction endonuclease digestion