Hydroxylamine Mutagenesis: Alternative Procedure via Elliot Altman
1. Mix 3.0 mls phosphate-EDTA buffer
4.5 mls sterile H20
6.0 mls hydroxylamine solution
1.5 mls P22 phage
2. Remove 50 ul into 5 ml of ice cold LBSE, and store at 4oC. This will serve as 0 time control point.
3. Incubate at 37oC.
Remove additional 50 ul aliquots into 5 ml of ice cold LBSE, storing at 4oC, at 8-10 hrs, 16-20 hrs, 24-30 hrs, and 32-36 hrs. Try to stay as close to 8 hr intervals as possible.
4. At 27 and 33 hrs, remove 7 mls of the sample and add this to 20 mls of ice cold LBSE in a 40 ml plastic centrifuge tube. Store at 4oC.
5. After the 33 hr sample is removed, centrifuge both the 27 and 33 hr samples in a SS-34 sorvall rotor at 17K rpm for 2 hrs at 4oC to pellet the phage.
6. At the end of the spin decant the supernatant and do a quick wash with ice cold LBSE.
7. Resuspend the pellets in 1 ml of ice cold LBSE on ice overnight with periodic vortexing.
8. Titer the 0 hr, 8-10 hr, 16-20 hr, 24-30 hr, and 32-36 hr 5 ml samples that were stored a 4oC. This will give you a killing curve.
Note - We have fould that the most critical factor in doing hydroxylamine mutagenesis is the preparation of the phosphate-EDTA buffer. If this buffer is prepared correctly (i.e.; it is truely at pH 6.0) then the 27 hour sample will yield 1-2% survival, while the 33 hour sample will yield 0.5-1% survival.
LBSE Phosphate EDTA buffer
10.0 g tryptone
5.0 g yeast extract 800 mls of dH2O with a stir bar
58.45 g NaCl 50.35 g KH2PO4 monobasic
0.37 g EDTA 22.64 g K2HPO4 dibasic
1.86 g EDTA
qs to 1000 mls
autoclave in 100 ml aliquots pH to 6.0
qs to 1000mls with dH2O
Hydroxylamine Solution (make this fresh)
1.12 ml 4 M NaOH
0.7 g NH2OH
qs to 10 mls with dH2O
Last Update: Thursday, 19-Jun-2014 12:03:03 PDT
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