Preparation of Tailspike (Older Method)

SDB 9/96


1. Growth

Inoculate 1 L superbroth with 10 ml of overnight culture (7136, su-). Shake at 250 rpm and 30ºC.


2. Infection

When cell density equals 4 x 108, infect at an m.o.i. of 5-10 (7). (Save 1-ml samples of each culture immediately before infection & 10 min after infection. Store on ice. Plate 0.1 ml of a 106 dilution of the uninfected sample & 0.1 ml of a 104 dilution of the infected sample.)


3. Harvest cells

Three hours after infection, chill flasks on ice (about 15 min). Perform lysis test: add one drop chloroform to a 1-ml sample of each culture & monitor for lysis. Pellet cells for 5 min at 5 K. Use clear (non-autoclavable) GSA bottles. Add bleach to supernatants.


4. Storage

To each bottle add 10 ml of 62.5 mM Tris (pH 6.8), 1 mM EDTA, and 40 mM octylglucoside. (Prepare a total of 50 ml of TE+OGP.) Resuspend pellets by shaking at low speed in the cold room. Pool into one bottle, rinse with remaining 10 ml, & store at -35ºC.


5. Lysis

Thaw frozen cells by sonication (twice for 15 min in ultrasonic cleaner in cold room). Shake at 30ºC & 250 rpm for 20 min.


6. DNase treatment

Add MgSO4 to 20 mM and DNase to 20 ug/ml. Continue shaking for 30 min. Measure volume (=1X). Save 100-200 ul for SDS-PAGE (L=lysate).


7. Pellet debris

20 min, 10 K, 4ºC. Carefully decant supernatant (pellet may be loose) into clean TI45 tubes on ice. Save 100-200 ul (LS= lysate supernatant). Add 10 ml TE+OGP to pellet. Resuspend by shaking at low speed in cold room.


8. Lysis & DNase treatment (second round)

Freeze suspension in liquid nitrogen. Thaw in water bath with shaking (30ºC). Add MgSO4 and DNase. Shake for 30 min at 250 rpm & 30ºC. Pellet debris for 10 min at 10 K (4ºC). Save 100-200 ul supernatant at 1X (NS=nitrogen supernatant). Decant supernatant into TI45 tube containing the lysate supernatant. Fill tube to neck with TE buffer, cover & weigh. Bring second tube to same weight. Resuspend pellet in 5 ml TE buffer using Dounce homogenizer. Save 100-200 ul at 1X (NP=nitrogen pellet).


9. Ultracentrifugation

1 h, 35 K (TI45 rotor), 4ºC. Transfer supernatant to graduated cylinder & record volume. Transfer to clear GSA bottle. Save 100-200 ul (35KS=35 K supernatant).


10. Salting out

Add AmSO4 (40% = 242 g/L). Dissolve salt by shaking at low speed in cold room. Incubate at 4ºC overnight.