Introduction of methionine auxotrophy into an Escherichia coli strain

1. Preparation of P1vir lysates of the donor auxotroph

Inoculation of 5 ml Luria Bertani (LB) medium with a single colony of the donor strain at 37 degrees overnight

Inoculation of 0.05 ml of the overnight culture in 5 ml of LB medium containing 0.2% (w/v) glucose and 5 mM calcium chloride.

• Incubate for 30 minutes at 37 degrees (with aeration).
• Add 0.1 ml of P1vir lysate (5 x 10 +08 phages/ml).
• Shake and rotate at 37 degrees for 1.5-3 h until the cells lyse.
• Add 0.1 ml of chloroform and vortex.
• Spin down cell debris at 4500g for 10 min.
• Transfer the supernatant to a sterile, screw-capped tube. Add 0.1 ml of chloroform and mix.
• Store the lysates at 4 degree.

1. Transduction to the recipient strain using P1vir lysate.

Inoculate 5 ml of LB medium with the recipient strain and shake at 37 degree overnight.

Centrifuge the overnight culture at 1500g for 10 min and resuspend the cell pellet in 2.5 ml of 10 magnesium sulfate containing 5 mM of calcium chloride.

To five test tubes add recipient cells and P1vir grown on the donor strain as follows:

Test tube	Cells	P1vir lysate
1 2 3 4 5	0.1 ml 0.1 ml 0.1 ml 0.1 ml none	none 10 ul 50 ul 0.1 ml 0.1 ml

1. Incubate the 5 test tubes for 30 min at 30 degree without shaking
2. Add equal volume of 1M sodium citrate to each tube and mix
3. Add 2.5 ml of appropriate molten top agar to each tube and plate on selective media

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Maciej Kozak's PPS'97 project
Protein engineering and its role in solving the phase problem
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