Rensselaer/12 September 2008

Goal: to purify the ZntA promoter and the dephosphorylated mRFP insert, and to quantify the amount of the resulting purifications as well as phosphorylated mRFP and the Fe promoter.

Purification:

1. Added 100 uL of Membrane Binding solution to 100 uL ZntA promotor and 200 uL of Membrane Binding Solution to 200 uL deP mRFP insert. Pipetted to mix and incubated for 1 minute. 2. Transferred mixtures to respective wash columns and centrifuged at 16,000g for 1 minute. Discarded Wash in collection column. 3. Added 700 uL of Wash solution to each wash column and centrifuged 16,000g (14,000 rpm) for 1 minute. Wash was then discarded and 500 additional uL of the Wash solution were added and centrifuged at 16,000g for 5 minutes. Wash was again discarded, and empty columns were centrifuged again for 1 minute at 16,000g to evaporate remaining ethanol from wash columns. 4. Wash columns were transferred to 1.5 mL centrifuge tubes. 50 uL of nuclease free water was added to each column followed by 1 minute of incubation. The tubes were the centrifuged for 16,000g for 1 minute.

Results of nanodrop readings:*

ZntA promoter. . . 7.6 ng/uL

Fe promoter. . . 29.3 ng/uL

mRFP insert. . . 31.4 ng/uL

dP mRFP insert. . . 30.3 ng/uL


 * The ZntA result is the average of three readings: 7.0, 8.0, and 7.8 ng/uL