DNA Restriction Digestion Analysis - G-Biosciences
methylates the restriction endonuclease site on the genomic DNA preventing digestion The combined role of these two enzymes is known as the restriction:modification system Each restriction endonuclease recognizes a specific se quence of nucleotides, normally ranging from 4 -8 base pairs in length The shorter the recognition sequence the more
New LC-MS Workflows Enabled by Efficient In-Line Digestion
digestion using sequence specific proteases is time consuming due to slow kinetics and subject to contamination from external sources, making it unreliable Typical digestion protocols require from 1:100 to 1:20 (w/w) trypsin to protein ratios, with digestion times ranging from 1 hour to overnight Upon analysis, if a significant amount of
Restriction Digestion and Analysis of Lambda DNA Kit
digestion patterns, analyze the migration distances, and determine the sizes of unknown DNA fragments Restriction enzymes were a catalyst for the molecular biology revolution, and now hundreds of such enzymes are known In this investigation, the restriction enzymes EcoRI, PstI, and HindIII will be used to digest bacteriophage lambda DNA
Assembly of Restriction Enzyme Digestions
15 minutes or less) and direct digestion in GoTaq® Green Master Mix or PCR Master Mix (1,2) The results are shown in Table 1 A protocol for rapid digestion is provided in Section 6 A, and a protocol for direct digestion of a PCR product is provided in Section 6 B Table 1 Compatibility of Restriction Enzymes With Rapid Digestion or Direct
DNA Scissors: Introduction to Restriction Enzymes Objectives
DNA fragments generated by restriction digestion can be put back together with the enzyme DNA ligase, which forms phosphodiester bonds between the 5' and 3' ends of nucleotides As you might expect, any blunt ended DNA can be ligated to any other blunt ended DNA without regard to the sequence of the two molecules Restriction fragments with single-
Enterokinase (EK)
EK digestion protocol Enterokinase is a specific protease that cleaves after lysine at its cleavage site Asp-Asp-Asp-Asp-Lys Bovine enterokinase light chain produced using bacterial system Unlike other site-specific proteases that cut within the recognition sequences leaving extra amino acids in the cleaved peptide/protein
Cleavage Close to the End of DNA Fragments (oligonucleotides)
Oligo Sequence Chain Length Cleavage 2 hr: 20 hr AccI: GGTCGACC CGGTCGACCG CCGGTCGACCGG 8 10 12 0 0 0 : 0 0 0 AflIII: CACATGTG CCACATGTGG CCCACATGTGGG 8 10 12 0 >90 >90 : 0 >90 >90 AscI: GGCGCGCC AGGCGCGCCT TTGGCGCGCCAA 8 10 12 >90 >90 >90 >90 >90 >90 AvaI: CCCCGGGG CCCCCGGGGG TCCCCCGGGGGA 8 10 12 50 >90 >90 >90 >90 >90
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