FPLC Purification of Oligos

1. First, wash the Mono Q column w/ water (since it should be in 20% EtOH)

-On FPLC, press “manual”, the “step forward”

-Set valve to 1.3, the step forward to Wash A.B. Set to Wash A.B. to 1.1

-This washes the pumps w/ water @ 1 mL/min for each pump. Right now, nothing is flowing through the column

-When done with wash, set to Valve 1.1, the 50% B (still washing with water; both lines in water). Setting the Valve to 1.1 means stuff is now flowing through the column. Wash for several minutes @ 0.1 mL/min to avoid back pressure from EtOH on the column, then bring the speed to 0.5 mL/min for several minutes.

While washing column with water, use a 3 mL syringe to inject 3 mL of water through the injection loop to clean out the loop.

2. On the Frac-100:

-Set on mL

-set “wait”= 0.3

-set “fraction size”= 0.5 mL

-set “delay”= 0.3

3. When done washing column with water, hit “End”. Remove A & B lines from water, and place in Buffers A & B.

-Buffer A= 20 mM NaOH pH 12.0

-Buffer B= 20 mM NaOH pH 12 1.5 M NaCl

4. Wash pumps with buffers A and B

-Press manual, set Valve to 1.3, the set Wash A.B. to 1.1

-Once washing is completed, set Valve to 1.1, 0% B @ 1 mL/min

-Then set to 100% B @ 1 mL/min

-Then set to 0%B again @ 1 mL/min

-You want to make sure you clean the column and then run enough buffer A through so that the Mono Q column is equilibrated in Buffer A

5. While the column is being equilibrated in Buffer A, prepare the oligo that is going to be purified.

-Dissolve a vial of oligo in 550 μL Buffer A

-Vortex the vial like crazy

-Heat the vial @ 90˚C for several minutes

-Put vial on ice for 2 min

-Spin down for 5 min

6. On the OD meter, set to 2 OD once the column is equilibrated in Buffer A. Then hit Auto-Zero.

7. Make sure pens are connected to chart recorder. Set chart speed to 20. Also, fill up carousel with enough tubes.

8. Load 3 mL syringe with 550 μL prepared olio, and inject into the loop. Then Run Method #8.

9. The end of method 8 re-equilibrates the Mono Q column with Buffer A, so you can do another injection right away.

10. When finished with purification, wash pumps and then the column with water. Finally, wash the pumps and then the column in 20% EtOH.