New England Biolabs, C1010S, NEB® Cloning Competent E. coli Sampler

Not sure which cloning strain to choose? The NEB Cloning Competent Related Categories Competent Cells,, Cloning Competent Cell Strains Applications Transformation FAQ Q: Why did Synthetic Biologist Chris Voigt of MIT choose NEB 10-beta for DNA assembly and cloning? A: Can a living cell perform computation? By encoding circuits in DNA and transforming them into a living bacterial host, organisms can be manipulated to process environmental cues and have altered biological function. This repurposing of biological function is the cornerstone of synthetic biology, and is transforming markets such as biofuels and the food industry by engineering novel organisms designed for manufacture or biological sensing. Accomplishing this requires reliable assembly of large DNA constructs with specific arrangements of DNA parts, and a well-characterized organism in which to put them. The design of large DNA constructs is complex and assembly can be a formidable challenge. Chris Voigt, a synthetic biologist at the Broad Institute, and his team developed a computational environment (Cello) to standardize design of these assemblies based on user-supplied parameters and constraint. As Voigt and his team point out, the behavior of genetic circuits is dependent on many parts whose function can vary depending on genetic context, strain and growth conditions. When it came to selecting a competent cell strain, they chose NEB 10-beta Competent E. coli. Why was NEB 10-beta was chosen for Cello? Reduced recombination from RecA1 mutation Ability to clone and propagate large plasmids High transformation efficiency Better growth on M9 + glucose than some other strains Resistant to phage T1 Defined genotype Already being used in many labs around the world Q: How should fragments be prepared for assembly using NEBuilder HiFi? A: Fragments can be prepared by the following methods: PCR-generated fragments can be cleaned-up by using Monarch PCR column or Exo-CIP Rapid PCR Cleanup Kit if amplicon purity is greater than 95%. If plasmid DNA was used as template during PCR, it can be removed by DpnI treatment if necessary. If multi-bands are observed, we recommend optimizing the PCR. If this is not possible gel purification is recommended. Gel extraction can introduce guanidine thiocynate (from the dissolving buffer) that can reduce the efficiency of the assembly reaction. To minimize this contamination, trim the gel slice so that a smaller amount of gel dissolving buffer can be used. Restriction enzyme digestion of a plasmid can be performed followed by heat-inactivation or column purification. Commercially ordered fragments can be re-suspended in nuclease-free water or TE buffer and directly used in the assembly reaction.