lambdaMGU2

Starting and Amplifying ATCC Bacteriophage Lambda Clones and Vectors

1. Make fresh plating bacteria.  Grow E. coli host strain overnight or at least to A₆₀₀ = 0.4 in medium containing 0.2% maltose (to give higher titers).
2. Spin down cells in a low speed centrifuge.  Resuspend in 0.4 volumes 10 mM MgSO₄ or SM buffer.  Store at 4°C.
3. Pipette 100 µL of the host suspension to a sterile test tube. Add 3 mL. of warm (50°C) LB lambda top agar (see below) containing 0.2% maltose and mix gently.  Pour onto plates.  Allow the plates to solidify.
4. Open vial according to instructions. Asceptically add 0.3 to 0.4 mL of liquid medium (SM or 10 mM MgSO₄) to the freeze-dried pellet and mix well. 
5. Spot a loopful or two on the lawn of the freshly poured bacteria.
6. Incubate overnight at 37°C.  Fresh plates give larger plaques.
7. Cut plaques out of agar and add them to 0.5 mL of 10 mM MgSO₄ or SM buffer and store at 4°C overnight.
8. Add 100 µL phage dilution to 100 µL prepared plating bacteria and mix gently.  Incubate in a 37°C water bath for 20 minutes to allow phage to adsorb.
9. Add 3 mL. LB lambda top agar (see below) containing 0.2% maltose and mix gently.  Pour onto plates.  Incubate overnight at 37°C.  Fresh plates give larger plaques.
10. Invert open plate over a chloroform-saturated adsorbent paper for 10 minutes. 
11. Add 7.5 mL of 10 mM MgSO₄ or SM buffer to the plate and allow to stand at room temp for 1 hour or in 4°C overnight.
12. Collect and save the liquid on the plate. This should be a high titer lysate. Add a few drops of chloroform if its going to be stored for more than a few days.

For Cre-lox conversion proecedures, please refer to the following reference: Gene (Amst.) 120: 135-141, 1992.

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