Hydroxylamine mutagenesis of phage
(Ning 2-88; supercedes "#8")
A. phage lysate preparation
1. Grow overnight culture of donor strain in 5 ml of NB.
2. Innoculate 3 ml of overnight culture into 60 ml of NB for about 6 hours to be just dense.
3. Add 240 ml of P22 broth containing 107-108 pfu/ml of LT2 grown P22 phage. Incubate the infected cells about 14 hours.
4. Spin down cells in GSA rotor at 4K rpm for 40 minutes.
5. Carefully decant supernatant into clean centrifuge bottles, sample lysate and titer on NB plate (should be 1010 pfu/ml or more). Spin down phage in SS-34 rotor at 17K rpm for 1.5 hours.
7. Add a total of 3 ml of T2 buffer to the phage pellet (concentrate phage 100x). Let the pellet soak overnight at 4oC, then gently resuspend the pellet.
8. Centrifuge concentrated lysate in SS-34 rotor at 7K rpm for 10 minutes.
9. Carefully decant supernatant into sterile screw-cap tubes, add chloroform, and vortex.
For large preps of phage:
1. 500 ml P22 phage broth in 2 liter flask.
2. use 1/10 as much phage (6x105/ml) in the broth.
3. innoculate with 10 ml o/n.
4. grow 6-8 hr (not o/n).
5. spin out cells (with or without CHCl3) 6K 30'.
6. add NaCl to phage to 5 M.
7. sit o/n 4oC, spin again 6K 30'.
8. add 70g PEG6000 (now called 8000) per liter.
9. sit on ice o/n with slow stirring, then spin out phage.
B. Hydroxylamine mutagenesis
1. Mutagenize 3 ml of concentrated phage lysate as follows:
1 ml concentrated phage lysates
2 ml 0.45 M potassium buffer, pH 5,5. plus 5 mM EGTA (titrate with HCl)
2 ml hydroxylamine solution (0.35 g hydroxylamine hydrochloride, 4.44 ml sterile water, 0.56 ml 4 M NaOH)
2. Titer phage lysate at time zero.
3. Incubate mutagenesis mixture at 37oC in incubator with agitation.
4. Titer the mutagenesis mixture at about 8 hour intervals. Plot the titer to time of mutagenesis ( on semi-log paper) to estimate the time of harvest. Phage should be harvested at about 1.5% to 0.1% of survival.
5. At the expected time, remove half of the mutagenesis mixture, titer the lysate, then centrifuge in SS-34 rotor at 17K for 2 hours to harvest mutagenized phage. Let the rest of the lysate continue mutagenesis which can be used if the first half is not mutagenized enough.
6. Carefully decant the supernatant and drain all liquid out of the tube.
7. Add half volume of T2 buffer. Let the pellet soak overnight at room temperature; then gently resuspend the pellet.
8. If you want to keep the mutagenized phage for a long period of time, the phage can be washed once, and resuspended in T2 buffer.
Ning says: For 1% survival will get 10% null matation. Roof says: for 1% get one mutation per 40 Kb. Ning says: ts are >1/10 the number of nulls. Ning adds that to get ts and other missense a light muagenesis (1%) is much better than a heavy one (0.1%), with heavy you get only nulls.
Last Update: Thursday, 19-Jun-2014 12:03:44 PDT
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