Rensselaer/11 September 2008

Purification:

1. Added X uL of Membrane Binding solution to X uL Fe promotor and X uL of Membrane Binding Solution to X uL 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.

Retrieval of insert by gel purification:

approximately 25 uL of DNA was inserted into gel wells. The Fe promoter was put in wells 1-4 and the mRFP insert was placed in wells 5-8. A fragment ladder was pipetted into a reference well in the top center of the gel. The gel was run for about 25 minutes before the fragments reached the extraction wells in the center of the gels. 25 uL of the extraction wells were removed every minute from the wells until most of the DNA had been obtained. (Note: Be careful of using the UV while performing this experiment as the light will evaporate the water that has been set in the extraction wells!) The ZntA promoter was first extracted around 25 minutes, and the mRFP insert was extracted around 35 minutes time.