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TRANSFORMATIONS (from homegrown TOP10 cells)

From the Benfey Lab

Get cells from -80 ¿C freezer (one 50 µl tube per transformation). Thaw on ice.

Dilute ligation reaction to equal the volume of cells, at a final concentration of 1X KCM:

2.5 µl ligation reaction
10 µl 5X KCM buffer
37.5 µl water

Chill diluted ligation reaction on ice.

Combine entire diluted ligation reaction with cells. Incubate on ice for 30 min.

Heat shock at 42 ¿C for 1 min.

Add 950 µl LB broth. Incubate at 37¿C, shaking (200 rpm), for 60 min.

Centrifuge at 3000 rpm for 3 min.

Remove 900 µl of supernatant.

Resuspend pellet.

Plate cells on LB/amp/xgal plate.

5X KCM Buffer: 

0.5M KCl

0.15 M CaCl2

0.25 M MgCl2 

Filter sterilize through 0.2 uM filter. Store at -20.

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