Flash content link
https://vlab.amrita.edu/repo/BIOTECH/MOL/Preparation_stocks_%20bacteriophage_lambda_plate_lysis_and_elution/index.swf
Objectives required
Procedure
- Prepare infected cultures of bacteria by adding bacteriophage suspension. (Note:- For a 10-cm diameter Petri dish: Mix 105 pfu of bacteriophage. For a 15-cm Petri dish: Mix 2 x 105 pfu with 0.2ml of plating cells).
- Add the infected culture into molten top agarose (3ml for 10-cm plate or 7ml for 15-cm plate).
- Mix well the contents in the tube by vortexing or tapping.
- Pour the mixed contents into a labeled agar plate without making air bubbles. Then swirl the plate for uniform distribution of bacteria and top agarose in it.
- Incubate the plate (without inversion) for 12-19hrs at 37°C.
- Remove the plate from the incubator and add SM buffer in to it (5ml for 10-cm plate or 10ml for 15-cm plate).
- Store the plates as such on a shaking platform at 4°C for several hrs.
- Transfer the entire possible amount of SM buffer from the plate into a sterile screw- or snap-cap polypropylene tube using a Pasteur pipette.
- Again add 1ml of fresh SM buffer to the same plate, swirl it and store in a tilted position for 15 minutes to allow entire buffer to drain into one area.
- Remove the SM buffer and combine it with first harvest.
- Add 0.1ml of chloroform to the tube containing solution and vortex the tube and then remove the bacterial debris by centrifugation at 4000g for 10 minutes at 4°C.
- Transfer the supernatant obtained to a fresh polypropylene tube.
- Add 1 drop of chloroform to the tube and store the resulting bacteriophage plate stocks at 4°C.
