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The template DNA must be eliminated by enzymatic digestion with a restriction enzyme such as DpnI, which is specific for methylated DNA. All DNA produced from most Escherichia coli strains would be methylated; the template plasmid that is biosynthesized in E. coli will, therefore, be digested, while the mutated plasmid, which is generated in ...
New England Biolabs (NEB) is an American life sciences company which produces and supplies recombinant and native enzyme reagents for life science research. [2] It also provides products and services supporting genome editing , synthetic biology and next-generation sequencing . [ 3 ]
In molecular biology, the DpnII restriction endonuclease family is a family of restriction endonucleases which includes DpnII from Diplococcus pneumoniae.These enzymes recognise the double-stranded DNA unmethylated sequence GATC and cleave before G-1, where it encompasses the full length of the protein.
In order to detect multiple mutations in the same fragment, post-PCR clean-up must be done before Surveyor nuclease digestion. Detection of multiple mismatches can also be improved by increasing time and amount of surveyor nuclease in the reaction, but this also increases the background due to non-specific cleavage.
Restriction digest is most commonly used as part of the process of the molecular cloning of DNA fragment into a vector (such as a cloning vector or an expression vector).The vector typically contains a multiple cloning site where many restriction site may be found, and a foreign piece of DNA may be inserted into the vector by first cutting the restriction sites in the vector as well the DNA ...
Usually, the in-gel digestion is run as an overnight process. For the use of trypsin as protease and a temperature of 37 °C the time of incubation found in most protocols is 12-15 h. However, experiments about the duration of the digestion process showed that after 3 h there is enough material for successful mass spectrometric analysis. [38]
Micrococcal nuclease (EC 3.1.31.1, S7 Nuclease, MNase, spleen endonuclease, thermonuclease, nuclease T, micrococcal endonuclease, nuclease T', staphylococcal nuclease, spleen phosphodiesterase, Staphylococcus aureus nuclease, Staphylococcus aureus nuclease B, ribonucleate (deoxynucleate) 3'-nucleotidohydrolase) is an endo-exonuclease that preferentially digests single-stranded nucleic acids.