Amplifying a Library

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 (grown in NZY containing 100 g/ml kanamycin).Shake vigorously at 37℃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 0.22g/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 g/ml.

6.Remove a 7-µl sample from each flask for the dilutions (next step),then continue shaking the flasks vigorously overnight.

7.Spread 200 µl of 10-4 and 10-5 serial dilutions of the two 7-µl samples from the previous step (diluent = NZY)on NZY plates containing 40g/ml tetracycline and 100 g/ml kanamycin.Count the colonies the next day.A colony count of ~100 on the 10-5 plates indicates ~5 ×1010 infected cells per culture (~5% of the input physical particles--a typical infectivity for fd-tet phage).The 1011 infected cells in both flasks are enough for every clone in a billion clone initial library to be represented by 100 infected cells in the amplification—presumably sufficient over-representation to preserve essentially all the diversity.

8.Purify virions from the cultures as in VirionPurification.DOC,omitting the detergent treatments.

9.In order to check the quality of the library,we extract ssDNA from a sample of the virions as in ssDNA.doc and sequence the degenerate ｉｎｓｅｒｔ region.The degenerate positions show up as an obvious series of superimposed small peaks.