DNA restriction enzyme digestion reaction
[Purpose]
Through this experiment, learn the basic principles and experimental techniques of DNA restriction enzyme digestion reaction.
[Principle of experiment]
1. Restriction enzymes can specifically bind to specific sites within or near a DNA sequence called restriction enzyme recognition sequence, and cut double-stranded DNA. It can be divided into three categories: Class I and Class III enzymes have both cleavage and modification (methylation) in the same protein molecule and depend on the presence of ATP. The class â… enzyme binds to the recognition site and randomly cuts the DNA not far from the recognition site, while the class â…¢ enzyme cleaves the DNA molecule at the recognition site and then dissociates from the substrate. Class â…¡ consists of two enzymes: one is a restriction endonuclease (restriction enzyme), which cuts a specific nucleotide sequence; the other is an independent methylase, which modifies the same recognition sequence. Most class â…¡ restriction enzymes recognize palindrome symmetric specific nucleotide sequences with a length of 4 to 6 nucleotides (such as EcoRI recognizes six nucleotide sequences: 5 "-G ↓ AATTC-3"), a few Enzymes recognize longer sequences or degenerate sequences. The type â…¡ enzyme cleavage site is in the recognition sequence, and some are cleaved at the axis of symmetry to produce blunt-end DNA fragments (such as Sma: 5 "-CCC ↓ GGG-3"); some are located on the side of the axis of symmetry , DNA fragments with single-stranded overhangs are called sticky ends. For example, EcoRI cuts the recognition sequence to produce two complementary sticky ends. 5 "... G ↓ AATTC ... 3" → 5 "... G AATTC ... 3"; 3 "... CTTAA ↑ G… 5" → 3 "… CTTAA G… 5"
2. DNA purity, buffer, temperature conditions and restriction enzymes all affect the activity of restriction enzymes. Most restriction enzymes are not affected by RNA or single-stranded DNA. When a small amount of contaminants enters the restriction enzyme storage solution, it will affect its further use. Therefore, when sucking the restriction enzyme, a new pipette tip must be used every time. If two restriction enzymes are used, care must be taken to provide their respective optimal salt concentrations. If both can use the same buffer, they can be hydrolyzed at the same time. If different salt concentrations are required, the restriction enzyme of low salt concentration must be used first, then the salt concentration is adjusted, and then hydrolyzed with restriction enzymes of high salt concentration. Alternatively, after the first digestion reaction is completed, extract with equal volume of phenol / chloroform, add 0.1 volume of 3mol / LNaAc and 2 volumes of absolute ethanol, mix and place in -70 ℃ low temperature refrigerator for 30 minutes. Centrifuge, dry, and re-dissolve in buffer to perform the second digestion reaction.
3. DNA restriction endonuclease digestion map, also known as the physical map of DNA, which consists of a series of multiple restriction endonuclease digestion sites determined by a series of positions, represented by a straight line or a circular pattern. There are many ways to construct a DNA restriction enzyme map. Generally, a variety of restriction enzymes are used in combination, and the restriction sites and relative positions of various enzymes are determined by comprehensively analyzing the sizes of the restriction fragments obtained by multiple enzymes and multiple enzymes in different combinations. The use value of the digestion map depends on its accuracy and precision. In the process of making the digestion map, in order to obtain a clear band electrophoresis pattern, the general amount of DNA is about 0.5-1μg. The optimal conditions for the restriction enzyme digestion reaction are different, and each enzyme has its corresponding digestion buffer and optimal reaction temperature (mostly 37 ℃). For plasmid DNA digestion reaction, the amount of restriction enzyme can be digested for 1-2 hours according to the standard system of 1μg DNA plus 1 unit of enzyme. But for complete enzymatic hydrolysis, the amount of enzyme must be increased, generally 2-3 times, or even more, and the reaction time should be appropriately extended.
[Experimental Instruments and Equipment]
Horizontal electrophoresis device, electrophoresis apparatus, desktop high-speed centrifuge, constant temperature water bath, micro pipette gun, microwave oven, ultraviolet transmission instrument, camera system
[Experimental Materials]
λDNA; recombinant pBS material or pUC19 plasmid; EcoRI enzyme and its digestion buffer; HindⅢ enzyme and its digestion buffer; Agarose.
[Experimental steps]
1. Number the clean, dry and sterilized eppendorf tubes (preferably 0.5ml), add 1μg of DNA with the corresponding restriction enzyme reaction 10 × buffer 2μl with a micro pipette, and then add double distilled water to make the total The volume is 19μl. After mixing the solution in the tube, add 1μl of enzyme solution. Flick the wall of the tube with your fingers to mix the solution. You can also use a microcentrifuge to shake the solution and concentrate the solution at the bottom of the tube.
2. After mixing the reaction system, place the eppendorf tube on a suitable support (such as a foamed plastic plate), and keep it in a 37 ° C water bath for 2-3 hours to complete the enzyme digestion reaction.
3. Add 2μl 0.1mol / L EDTA (pH 8.0) to each tube, mix well to stop the reaction, and store in the refrigerator for future use.
4. Prepare agarose gel to analyze the results of endonuclease digestion reaction
Prepare 1.5% agarose gel, mix 10μl of enzymolysis solution with 2μl of 6 × sample loading solution and mix and run electrophoresis. Keep the voltage at 60-80V, and the current at 40mA. After electrophoresis, stain with EB for 20-25min, and observe the result under ultraviolet light.
Intelligent Supporting Facilities
Automatic Curtain Open and Close,Automatic Curtain Slider,Automatic Curtains Ikea
Jilin Province Yuaoda Trading Co., Ltd , https://www.yuaodacn.com