Smith Lab,Division of Biological Sciences,University of Missouri
http://www.biosci.missouri.edu/smithgp/PhageDisplayWebsite/AmplifyingLibrary.doc
This section describes how to amplify a library with minimal danger of substantially reducing its diversity. Amplification is accomplished by infecting fresh cells with a portion of the library, growing the infected cells in large cultures, and isolating the phage secreted by the infected cells into the medium. They key to maintaining diversity is to ensure that the number of cells infected (before the cells have had a chance to replicate) is much larger than the number of clones in the primary library.
1. Inoculate two 1-liter culture flasks containing 100 ml terrific broth with 1 ml of an overnight culture of K91BluKan. Shake vigorously at 37º until the OD600of a 1/10 dilution reaches ~0.2 (late log phase).
2. Slow the shaking way down for 5 min to allow sheared F pili to regenerate.
3. Into each flask add ~1012 physical particles (~5 × 1010 TU)of the library to be amplified. The final concentration of physical particles (1010 virions/ml) is roughly comparable to the concentration of viable host cells. Continue slow shaking for 15 min.
4. Pour each culture into a pre-warmed 3-liter fernbach flask containing 1 liter of NZY supplemented with 0.22 mg/ml tetracycline; shake vigorously for 35 min at 37º.
5. To each flask add 1 ml 20 mg/ml tetracycline, bringing the concentration up to 18 mg/ml. If the library is in the f88-4 vector, also add 1 ml 1 M IPTG to induce expression of the recombinant (peptide-bearing) gene VIII.
6. Remove a 7-µl sample from each flask for the dilutions (next step), then continue shaking the flasks vigorously overnight.
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