Resuspend the keygen for everything manager 6.12 bacterial pellet in 200 l of ice-cold Alkaline lysis solution I by vigorous vortexing, and file H/temp/Protocols/m (1 of 8) 22:34:35 Molecular Cloning Protocol 2Print Version transfer the suspension to a microfuge tube.
To avoid producing air bubbles, mix the medium by swirling.
Protocol 12: Removal of Ethidium Bromide from nissan leopard repair manual 97 DNA by Extraction with Organic Solvents Ethidium bromide is removed from DNA by phase extraction with organic solvents.To prepare 2x YT medium, shake until the solutes have dissolved.Protocol 8: Purification of Plasmid DNA by Precipitation with Polyethylene Glycol Crude preparations of plasmid DNA are first treated with lithium chloride and RNase (to remove RNA).Remove all of the supernatant by gentle aspiration as described in Step.Take care with this step, as the pellet sometimes does not adhere tightly to the tube.TE 100 mM Tris-Cl (desired pH) 10 mM edta (pH.0) file H/temp/Protocols/m (5 of 8) 22:34:33 Molecular Cloning Protocol 1Print Version (10x Tris edta) Sterilize solutions by autoclaving for 20 minutes at 15 psi (1.05 kg/cm 2) on liquid cycle.Molecular Cloning from an essential laboratory resource into a new realm, one merging the previous prototype with a modern molecular e next generation.
Hold the flask in the boiling water for exactly 40 seconds.
Be as gentle as possible to minimize shearing of the liberated chromosomal DNA.
Through the viscous bacterial lysate by inverting the tube several times.
Dissolve the nucleic acids in 50 l of TE (pH.0) containing 20 g/ml DNase-free RNase A (pancreatic RNase).
File H/temp/Protocols/m (6 of 6) 22:34:41 Molecular Cloning Protocol 5Print Version Chapter 1, Protocol 5 Preparation of Plasmid DNA by Large-scale Boiling Lysis Plasmid DNA is isolated from large-scale (500 ml) bacterial cultures by treatment with Triton X-100 and lysozyme, followed by heating.Add to each tube.5 l of a solution containing.4 bromophenol blue if the samples are to be analyzed only by agarose gel electrophoresis or 2 l of 10 mM cresol red if the samples are to be analyzed both by PCR and.Collect the precipitated nucleic acids by centrifugation at maximum speed for 5 minutes at room temperature in a microfuge.Expanded authorship: chapters and protocols have been specifically commissioned from renowned experts at leading institutions.When this condensation drops on the agar/agarose surface, it allows bacterial colonies or bacteriophage plaques to spread and increases the chances of cross-contamination.
Use one of the methods described in Chapter 1, Protocol 1 or Chapter 1, Protocol 4 to prepare plasmid DNA from the 1-2-ml al"of bacterial culture set aside in Step.