Restriction endonuclease profiling of λ phage DNA
Restriction endonuclease profiling of λ phage DNA
Lambda phage is a commonly used biological material in molecular genetics research, and the enzymatically fragmented segments of its DNA are often used as standards for determining the molecular weight of DNA. Restriction endonucleases are a class of DNA hydrolases that recognize specific base sequences in double-stranded DNA molecules and are mainly found in prokaryotic cells. According to the difference of these enzymes, they can be named as type I, II and III respectively. Type Ⅰ and Ⅲ restriction endonuclease in the same protein molecule both modification (methylation) and ATP-dependent restriction (cleavage) activity, but can not degrade the DNA in specific sequences, so in its enzyme profiling and molecular cloning is rarely used, the main application is Ⅱ type of enzyme, because this type of enzyme can recognize a highly specific 4-6 nucleotide sequences, cut to produce the 5′ end of the prominent This type of enzyme recognizes highly specific 4-6 nucleotide sequences and produces either a complementary single-stranded end with a protruding 5′ end (sticky end) or a flat end without a protruding single-stranded end (or blunt end). Both types of ends can be ligated with T4 ligase. Although the efficiency of joining flat ends of DNA is lower than that of sticky ends, they are universally adaptable and have therefore become a common DNA joining strategy in molecular biology. Source: Laboratory Course in Genetics
Operation method
basic program
Principle
λDNA is a linear double-stranded DNA, on which EcoRⅠ has 5 cut points, resulting in 6 fragments, which can be separated by agarose gel electrophoresis. How to reconstruct these 6 fragments? Two kinds of restriction endonucleases can be applied to λDNA at the same time or successively, for example, the six bands separated from λDNA after EcoRⅠ cleavage can be eluted, and then these six fragments can be cleaved by Hind Ⅲ respectively. The results showed that there was no Hind Ⅲ cleavage site on fragment 1, while there were two Hind Ⅲ cleavage sites on fragment 2, and there were six Hind Ⅱ cleavage sites on the complete linear λDNA to produce seven fragments. A preliminary restriction endonuclease profile could be made based on the relationship between fragment size and cleavage sites. The most commonly used method to convert restriction endonuclease maps into gene alignment maps is Southernblot, in which the electrophoresed enzyme section is hybridized with a particular gene DNA or its RNA probe, and the gene is then located in a certain enzyme section.
Materials and Instruments
λPhage DNA Move (I) λDNA-EcoRⅠenzymatic reaction 1. Take a new sterile Eppendorf tube and add 16 μL of sterile deionized water, 2 μL of 10×EcoRⅠ buffer, 1 μL of EcoRⅠ enzyme, and finally 1 μL of λDNA (the total volume of reaction is 20 μL), this is a small amount of enzyme reaction system, which is mainly used for the identification of enzyme digestion. For the preparation of gene fragments, a large amount of enzymatic reaction system can be used, with a reaction volume of 50-100 μL, and the amount of various reagents added can be expanded accordingly. 2. Gently mix the reaction reagents in the Eppendorf tube, and terminate the reaction after 1~1.5h in a 37℃ water bath (according to the requirements of different enzyme specifications). 3. Remove the sample for agarose gel electrophoresis with appropriate concentration (taking into account the recovery of DNA from the gel, low melting point agarose can be used to make the gel). (ii) Recovery of DNA fragments from agarose gel 1. Carefully cut off the gel containing the DNA fragments to be recovered under a long-wave ultraviolet lamp and put it into an Eppendorf tube. 2. Gently crumble the gel in the Eppendorf tube, add 2-5 times the volume of TE buffer, keep warm at 65℃ for 10min, remove and cool to room temperature. 3. Take out the recovery tube, add an equal volume of saturated phenol and mix thoroughly, then centrifuged at 4000 × g for 10 min. 4. Take the aqueous phase in another Eppendorf tube, and then extract it once with an equal volume of chloroform-isoamyl alcohol (24:1) mixture. 5. After centrifugation at 4000×g for 10 min, the supernatant was transferred to another new Eppendorf tube, and 0.2 times the volume of 10moL/L ammonium acetate and 2 times the volume of cold ethanol were added, and the mixture was mixed well, and then left to stand at room temperature for 10 min. 6. Centrifuge the sample at 12000×g for 20min at 4℃, discard the supernatant and rinse it twice with 70% ethanol; bake the DNA sample at 65℃ for 5~10min until dry, and add appropriate amount of TE buffer or sterile deionized water to dissolve the DNA. 7. Take a small amount of dissolved DNA samples for agarose gel electrophoresis to detect the purity and concentration of the recovered DNA. 8. Recover the other 5 DNA fragments in the same way. (C) λDNA-Hind Ⅲ digestion reaction 1. Take a new sterile Eppendorf tube, add 13-15 μL of double-distilled water, 2 μL of 10×Hind Ⅲ buffer, 1 μL of Hind Ⅲ enzyme, and finally add 10 μL of a fragment of DNA recovered from λDNA-EcoR Ⅰ digestion. 5 Eppendorf tubes were taken to make Hind Ⅲ enzyme reaction system for the other 5 fragments. 2. Gently mix the enzymatic reaction system, and then place it in a 37℃ water bath for 1~1.5h to terminate the reaction. 3. Take an appropriate amount of sample for agarose gel electrophoresis analysis. At the same time, use λDNA/EcoRⅠ standard, λDNA/HindⅢ standard, λDNA/(EcoRⅠ+HindⅢ) standard control. (IV) λDNA/EcoRⅠ+HindⅢ double enzymatic reaction When the reaction conditions of the two enzymes are the same or similar, the double enzymatic reaction can be carried out with reference to the small amount enzymatic reaction. When the reaction conditions are different, for example, when the buffer NaCL concentration is too different, the enzyme activity will be inhibited and the enzymatic reaction should be carried out separately, which can be carried out in the order from low salt to high salt. If this method is not used, the ethanol precipitation method can be used to re-establish the reaction system by precipitating the DNA that has been degraded by one of the enzymes. Each restriction endonuclease has its own optimal reaction conditions, and generally speaking, the conditions of the calibrated enzyme unit are the standard conditions for the reaction. Each restriction endonuclease product often recommends different reaction conditions for the same enzyme, so it is recommended to follow the instructions of the product. In this experiment, the ethanol precipitation method was used, in which the DNA digested by one enzyme was precipitated first, and then the reaction system was rebuilt. 1. Take a new sterile Eppendorf tube, add 16 μL of sterile deionized water, 2 μL of 10×EcoRⅠ buffer, 1 μL of EcoRⅠ enzyme, and finally 1 μL of λDNA, and then gently mix well. 2. Place the sample solution in a 37℃ water bath and keep warm for 1~1.5h, then terminate the reaction. 3. Use ethanol precipitation method to precipitate the DNA that has been digested by one enzyme. 4. Add 17μL of sterile deionized water to dissolve the precipitated and dried DNA. 5. Add 2 μL of 10×Hind Ⅲ buffer and 1 μL of Hind Ⅲ enzyme to the DNA sample solution sequentially. 6. After gently mixing, keep the reaction at 37℃ for 1~1.5h and then terminate the reaction. 7. Take an appropriate amount of digested sample for agarose gel electrophoresis. At the same time, λDNA/EcoRⅠ standard, λDNA/HindⅢ standard and λDNA/EcoRⅠ+HindⅢ standard were used as control. Caveat 1. The amount of DNA and enzyme added in the procedure can be adjusted according to different batches of products, but the total volume of reaction remains unchanged. 2. Generally, the DNA sample is added last to avoid the contamination of reagents caused by improper operation. 3. Replace the tip of the micro-sampler each time you add a sample (except for adding water first) to avoid cross contamination. 4. When the sample is added to the reaction tube, the Eppendorf tube should be capped to prevent water vapor from entering the tube when it is kept warm. For more product details, please visit Aladdin Scientific website.
λDNA EcoRⅠ standard λDNA HindⅢ standard λDNA(EcoRⅠ+HindⅢ) standard Restriction endonuclease EcoRⅠand HindⅢ Restriction endonuclease reaction 10×buffer Agarose gel electrophoresis buffer Recovery of DNA in agarose gel system
Constant temperature water bath Oscillator Agarose gel electrophoresis device Sampling gun Centrifuge Gel electrophoresis imaging system Micropipettes and tips Eppendorf tubes