Rensselaer/10 August 2008

Promoter Group

Miniprep of Ligation Transformed Cultures using Promega Wizard Plus Miniprep kit.

Production of a Cleared Lysate

Harvest cells by spinning @ 4000 RPMs, 10', 4C.

Pour off supernatant.

Resuspend cell pellets in 250 ul Cell Resuspension Solution.

Transfer cells to 1.5 ml tubes.

Add 250 ul Cell Lysis Solution. Invert 4x to mix.

Incubate until cell resuspension clears (no more than 5').

Add 10 ul Alkaline Protease Solution. Invert 4x to mix. * Took a little longer than 5 minutes to add APS

Incubate @ RT for 5'.

Add 350 ul Neutralization Solution. Invert 4x to mix. * Ran out of neutralization solution, so prepped 8 samples not 10

Centrifuge max speed 10' @ RT.

Plasmid DNA Isolation

Transfer cleared lysate to Spin Column.

Centrifuge max speed 1' @ RT. Discard flowthrough.

Add 750 ul Column Wash Solution. * Not enough CWS, prepped just 6 samples.

Centrifuge max speed 1' @ RT. Discard flowthrough.

Add 250 ul Column Wash Solution.

Centrifuge max speed 2' @ RT.

Transfer column to new 1.5 ml tube.

Add 100 ul H2O.

Centrifuge max speed 1' @ RT.

Store @ -20C.