Mutagenesis - Bacteriophage P22 with Hydroxylamine
Jeff Lawrence (X05)
1. Prepare 100 mL bacteriophage P22 lysate, removing cell debris by centrifugation.
2. Concentrate bacteriophage by centrifugation at 17 krpm for 150 min, or PEG precipitation.
3. Resuspend bacteriophage in 1 mL T2 buffer to form concentrated bacteriophage.
4. Prepare phosphate buffer as:
0.45 M K2PO4, pH 5.5
5 mM EGTA
5. Prepare hydroxylamine solution as:
0.35 g hydroxylamine hydrochloride
4.44 mL sterile water
0.56 mL 4 M NaOH
1 mL concentrated bacteriophage
2 mL phosphate buffer
2 mL hydroxylamine solution
7. Titer bacteriophage at time zero.
8. Incubate mixture at 37°. Titer mixture every 8 hours. Plot log(titer) vs. time to estimate time to harvest. Mutagenesis should be terminated when bacteriophage titer drops to 1% to 1.5% of its original titer. This will lead to 10% null mutation, or 1 mutation per 40 kb.
9. At the expected harvest time, remove 50% of the mixture and centrifuge for 150 min at 17 krpm. Let the remaining bacteriophage continue with mutagenesis in case the first harvest was incorrect.
10. Decant the supernatant and drain all liquid out of the tube. Resuspend in 1 mL T2 buffer. Let the pellet soak overnight at room temperature to resuspend.
11. If the lysate is to be stored, resuspend in a large volume of T2 and repeat centrifugation. This will rinse more of the hydroxylamine away.
Last Update: Thursday, 19-Jun-2014 11:54:46 PDT
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