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One is not normally required to serially dilute E. coli cultures for spectrophotometric measurements, at least in the experiments where the OD value is important. For most protein expression work, the consensus is to start the induction at OD 0.6, and for most chemically competent cells, the optimal OD ranges from 0.3 to 0.8 depending on the competency ...


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E.coli can do that and in fact does this a lot in a commonly used bacterial medium: The popular LB Medium. This is composed of three components: Tryptone, a peptide mix made by digesting milk casein with the peptidase Trypsin (10g/l) Yeast extract, made by autolysis of the cells (5g/l) Sodium chloride (10g/l) The original formulation of Bertani from ...


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The root cause turned out to be a lying spec. Using a different one, it was revealed that there was next to no template DNA in the initial amplification. No DNA, no good plasmids.


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In general, there is no need for a spacing between stop codon and terminator. They can be right next to each other. When transcribed, transcription terminator forms a hairpin-like structure which interacts with the RNA polymerase to stop the transcription. Usually, the upstream sequence will not interact with the terminator and it will be transcribed ...


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You should dilute (not necessarily serially dilute) if: You are exceeding the concentration range detectable by the machine Scattering starts overwhelming absorption (at high cell densities scattering will overwhelm absorbance). In such cases you can use a turbidimeter. As such there is no issue of the law not holding true at certain limits of ...


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Reading the optical density of a log phase microbial culture at a fixed wavelength approximates the Beer-Lambert law where A = ecl. This relationship is not linear as the Absorbance approaches 1.0 so at higher cell densities you will eventually underestimate the cell density of the culture. Likewise the law is not accurate below an Absorbance below ~ 0.05, ...



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