Assemble all components, perform the lower-temperature digest first, and then digest at the higher temperature. If you must work with two enzymes with different optimum temperatures, you can use the sequential digest method. Some enzymes work best below 37☌, like SmaI (25☌) and CspI (30☌). The majority of restriction enzymes work best at 37☌, but those isolated from thermophilic bacteria require higher temperatures for maximal activity (e.g., BstXI and BstZI work best at 50☌). Restriction Digestion: Other Considerationsĭo both enzymes work at the same temperature? Glycerol concentrations >5% may lead to enzyme star activity, which means the enzyme will lose sequence specificity and may produce random cuts in the DNA. Do not exceed 5% glycerol in final digest with the two enzymes. Remember, restriction enzymes are commonly stabilized in 50% glycerol solution. Simply add 1µl of the second restriction enzyme and adjust the amount of water used to maintain a 20µl reaction. If both restriction enzymes work in the same restriction enzyme buffer, the reaction is straightforward. Preparing an insert for transfer from one vector to another usually requires digestion with two different REs. Add 4µl of 6X Blue/Orange Loading Dye and analyze digested DNA by gel electrophoresis. Incubate at the appropriate temperature and the appropriate time for the enzyme. Mix by pipetting and collect the contents at the bottom of the tube. For a digestion with a single RE the reaction is very simple: Restriction digestion is one of the most common reactions performed in molecular biology. Many restriction enzymes will not cut DNA that is methylated on one or both strands of the recognition site, while some require substrate methylation. Type II REs cleave double-stranded DNA (dsDNA) at specific sites within or adjacent to their recognition sequences. Restriction endonucleases (RE), also referred to as restriction enzymes, are proteins that recognize short, specific (often palindromic) DNA sequences. Many strategies have been employed to do partial digests including decreasing reaction temperature, using a non-optimal buffer, and decreasing units of enzyme. You can manipulate the restriction digest conditions such that you will digest only a subset of sites.
Under normal restriction digest conditions, the enzyme is in excess so that all recognition sites in the plasmid can be cleaved. The presence of the same restriction enzyme recognition site in the insert and the multiple cloning region does not necessarily preclude use of that restriction site in a subcloning strategy. Subcloning Strategy: Common Restriction Sites with Partial Digests Controlling Cut Frequency in Restriction Digestion Partial Restriction Digestion can be performed. Having a restriction site in both the multiple cloning region and the insert does not exclude the use of this site for subcloning. These nicks will be repaired in the bacteria upon transformation. The vector has been dephosphorylated so the second bond will not be formed in vitro (indicated by the OH). coli, and those more experienced researchers a broad-ranging, proven array of successful techniques.The T4 DNA Ligase will join the DNA by reforming the bond between the 5´-PO 4 coming from the insert and the 3´-OH of the vector. coli Plasmid Vectors offers those new to the field a basic guide to the use of plasmid vectors in the cloning host E. Each fully tested protocol is described in step-by-step detail by an established expert in the field and includes an introduction outlining the principles behind the technique, lists of the necessary equipment and reagents, tips on troubleshooting and avoiding known pitfalls and, where needed, a discussion of the interpretation and use of the results.Ĭomprehensive and highly practical, E. Also presented are methods for common downstream applications, such as mutagenesis, expression of recombinant proteins and RNA transcripts, and uses of reporter genes. They also include protocols for the construction and screening of libraries, as well as specific techniques for specialized cloning vehicles, such as cosmids, bacterial artificial chromosomes, l vectors, and phagemids. The authors describe readily reproducible methods for cloning DNA into plasmid vectors, transforming plasmids into E. coli Plasmid Vectors, experienced bench researchers describe their proven techniques for the manipulation of recombinant plasmids utilizing this popular bacterial host.
coli, constitutes one of the fundamental methodologies of molecular biotechnology. Manipulation of recombinant DNA, which is almost exclusively performed using the host E.
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