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 40 mg/ml tetracycline and 100 mg/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 steps 2–4, 6–7 and 9–20 of VirionPurification.DOC, omitting the detergent treatment.
9. We usually sequence a sample of the phage as described in sequencing.doc to confirm the degenerate region of the coding sequence.
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