Rensselaer/24 August 2008

Promoter Group

Quick Screen of Fe Promoter Ligation Colony and J12006 Plasmid Culture

One of the j12006 cultures was not red (must have lost plasmid). quick screen only one j12006 culture.

Harvest cells @ 4000RPM 10' 4C. Discard supernatant.

Transfer cells to 1.5 ml tube.

Add 100 ul solnI + 2 mg/ml lysozyme.

Vortex 3-5''. Ice 10'.

Add 200 ul solnII. (4.65ml H2O, 250ul 20%SDS, 100ul 10M NaOH)

Vortex 2''. Ice 5'.

Add 150 ul soln III (3M KAc).

Vortex 3-5''. Ice 15'.

Microfuge max speed 10' 4C. Transfer supernatant to new 1.5 ml tubes.

Add 270 ul isopropanol. Dry ice 15'.

Microfuge max speed 10' 4C. Discard supernatant.

Add 100 ul H2O. Resuspend pellet.

Add 150 ul 5M LiCl. Ice 15'.

Microfuge max speed 10' 4C. Transfer supernatant to new 1.5 ml tubes.

Add 750ul 100% EtOH. Dry ice 15'.

Microfuge max speed 10' 4C. Discard supernatant.

Resuspend pellet in 50 ul H2O (estimate [DNA] to be .5 ug/ul-1 ug/ul)

Store DNAs @ -20C.