due 24 hours

Welcome!  

Experiment  II,  Day  6   September  29,  2016   (Working  individually)  

Transduction •  Using tubes from last week, VORTEX your culture and check

the O.D. 625nm (Blank 1st with LB), to be sure it has lysed. •  Add a drop of chloroform to your P1Cm c1-100, VORTEX. •  Transfer 1.0 ml to two 1.5 ml Eppendorf tubes (1.0 ml per

tube). •  Centrifuge for about 2 minutes,

–  BALANCE the tubes and put on the LID. •  Transfer supernatants to fresh microfuge tubes. •  Add another drop of chloroform, VORTEX. •  Centrifuge for 1 minute. •  Dilute the supernatant (from 1 tube) 10-1-10-7.

- Do you remember how? (20 µl of lysate to 180µl of diluent)

**SAVE the rest! PUT in rack for 4°C**

Titration of Phage •  Transfer 100 µl of fresh Escherichia coli KL227 in 3 empty 1.5 ml

Eppendorf tubes (100 µl per tube), and label the tubes 10-5, 10-6 & 10-7

•  Add 10 µL of 0.1 M CaCl2 to each tube (containing 100 µl of fresh E. coli KL227).

•  Add 100µl of each phage dilution (10-5, 10-6 & 10-7 ) to appropriate tube.

•  Incubate at 30°C for 10’. –  Label your plates (while waiting).

•  Only when you’re ready, retrieve the top agar from the water bath.

•  Add phage mixture (210 µl) to top agar tubes (roll to mix if necessary).

•  Pour onto plates.

•  When solid, incubate at 40°C.

Centrifuge

Transfer supernatant

*SAVE!*

KL227

Dilute Incubate

Pellet is waste

Lysate from last Tuesday

100 µl

1 ml

10-5

10-6

10-7

KL227

Dilute (10-1-10-7) Incubate

10-5

10-6

10-7

10-5 10-6 10-7

10-5 10-6 10-7

Top agar

•  Inoculate LB + rif medium with rifR mutant.

•  Incubate at 37°C.

Transduction 1.  VORTEX your culture and check the O.D. 625nm (Blank 1st with LB) 2.  Add a drop of chloroform to your P1Cm c1-100, VORTEX. 3.  Transfer 1.0 ml to two 1.5 ml Eppendorf tubes 4.  Centrifuge for 2 minutes at maximium speed

•  BALANCE the tubes and put on the LID. 5.  Transfer supernatants to fresh microfuge tubes, Add a drop of chloroform,

VORTEX, Centrifuge for 1 minute at max speed. 6.  Dilute the supernatant (from 1 tube) 10-1-10-7.

1.  20 µl of sample into 180µl of diluent 2.  **SAVE the rest! PUT in rack for 4°C [refrigerator in back]**

Titration of Phage 1. Transfer 100 µl of fresh Escherichia coli KL227 in 3 empty 1.5 ml

•  label the tubes 10-5, 10-6 & 10-7 •  Add 10 µL of 0.1 M CaCl2 to each tube

2. Add 100µl of each phage dilution (10-5, 10-6 & 10-7) to appropriate tube. 3. Incubate at 30°C for 10 minutes.

1.  While you wait…Label your plates 4. Only when you are ready, ONE PLATE AT A TIME

1.  Retrieve the top agar from the water bath. 2.  Add phage mixture (210 µl) to top agar tube, roll to mix if necessary. 3.  Pour onto plates; making sure to gently rock plate to cover the entire surface

5. When plates are fully solidified, incubate at 40°C.  

Clean-Up •  P1Cm c1-100 tube to 4°C rack.

•  Top agar plates to 40°C in back.

•  rifR mutants in LB to 37°C shaker in front.

•  Used titer plates and old rifR plates to biohazard waste.

•  Empty top agar and KL227 tubes to tube collection.

•  Microfuge tubes and Tip boxes on cart at the back.