Library Screening *

Phage Lambda Libraries *

Preparation of plating cultures *

Titering procedure *

Plating and Lifts *

 Library Screening

 

Phage Lambda Libraries

Preparation of plating cultures

1. Streak out the host cells over the plate using a flamed wire loop and incubate overnight at 37 C.

Note: Do not add antibiotic to the plate

Day one

2. Inoculate 50 ml of LB broth (containing 0.2 % maltose and 10 mM MgSO4 ) in a sterile Erlenmeyer flask with a single colony of the appropriate bacterial host.

Note: Do not add antibiotic to the plate

3. Grow overnight with shaking at 37 C for no more than 14 hours.

Day Two

  1. Spin down cells in a sterile conical tube for 10 minutes at 2000 rpm.

  2. Carefully decant the media off the cell pellet and gently resuspend the pellet in 25 ml of SM buffer.

  3. The cells may be stored for 2-3 days at 4 C

 

Titering procedure

  1. Make a few serial dilutions of the phage in SM buffer

  2. Add 200 ml of host cells per tube. Inoculate with diluted phage.

  3. Incubate at 37 C for 30 minutes.

  4. Add 3 ml 48oC top agar and plate on 100 mm NZY or LB plate

    5. Let plates harden at room temperature for 10 minutes.

    Incubate plates overnight at 37 C

  5. Count the number of plaques and determine the plaque forming unit (pfu/ml) concentration of your library based on dilutions.

Plating and Lifts

  1. Titer library

  2. Plate on large square (22cm) NZY or LB plates to 90000 pfu/plate with 1 ml host cells /plate and 30 ml top agar/plate. Incubation 30 minutes at 37 C

  3. Incubate at 37 C. Grow approximately 4 hours

  4. Refrigerate at least 30 min. at 4 C.

    5.  

    Note: Use forceps and wear gloves for the following steps.

  5. Transfer 1 minute on nitrocellulose or nylon membranes. Prick filter with needle through the membrane into the agar for orientation. For the second filter, transfer 3 minutes.

    6. A. Denature filter after lifting by submerging in (1.5M NaCl, 0.5M NaOH) for 5 minutes.

    B. Neutralize 5 minutes by submerging in ( 1.5M NaCl, 0.5M Tris-HCl pH8.0) C. Rinse 5 minutes by submerging in 2 x SSC

  6. Blot briefly on Whatmann 3 MM paper.

  7. Crosslink the DNA onto the filters for 8 minutes in a Lamina Flow Hood, or use a crosslinker.

  8. Store the agar stock plates of the transfers at 4oC to use after screening.