HPLC Procedure for DNA Purification

 

TURNING ON HPLC

  1. Press "sleep" buttons to wake up machines.
  2. Transfer PUMP B from 100% ACN jar to buffer B jar.
  3. Turn on lamp

            a. In "Analysis" screen, set "Lamp" to "1".

            b. Press "zero" button on SPD-10A.

  1. On computer:

            a. Open "CLASS VP Chromatography".

            b. Open "Corn HPLC".

            Normally the program is already opened in the computer.

 

WASHING THE COLUMN

  1. In "Analysis File" screen (1 in main menu), set "conc" to "100%", then press "PMP.ON" button (f1).  This flows 100% buffer B to the column, which takes 15-20 min to equilibrate.
  2. After pressure is stable, set "conc" to "10%".  This flows 10% buffer B and 90% buffer A. When the pressure is stable, the HPLC is ready for sample injection.

 

INJECTING THIOL-DNA

  1. In "sequence" screen (press 0 in main menu)

            a. Set 'sample #' = 1 - 1

            b. Set volume slightly less than sample volume, for example, 1 uL less

            c. Set 'file' = F0

  1. Put sample vial in position "1" on the sample rack.
  2. Injection:
    1. Press "single" on the computer, enter directory and file name
    2. On system controller (SCL-10A), press "run" button
  3. Wait for ~20 min for the DNA peak to show up on the computer screen.
  4. After collecting the DNA fraction, click on the "stop" button on the computer, and then press the "run" button on the system controller (SCL-10A) to stop HPLC in the middle of the run.
  5. If you have a second sample to purify, you need to wash the column by changing ̉conÓ to 100% and allowing the instrument to equilibrate.  Meanwhile, prepare your second sample for HPLC.  After a stable pressure has been reached, change the ̉concÓ to 10%, allow to stabilize and put your second vial in position ̉1Ó.  Repeat steps from # 1 in this section. 

 

SHUTTING DOWN

1. Turn off lamp: set "Lamp" to 0 in "analysis" screen.

2. Turn off pump:

            a. In "analysis" screen, press "PMP.OFF" button (f1).

            b. Wait until pump pressure drop to zero, then transfer pump B to 100% ACN.

            c. Turn pump on again, until pressure equilibrates (600-700 psi for ACN).

            d. Then turn pump off. Make sure pressure drops to zero.

3. Press the "sleep" button to put machines to sleep (DO NOT turn off the power).