HPLC Procedure for DNA
TURNING ON HPLC
- Press "sleep" buttons to
wake up machines.
- Transfer PUMP B from 100% ACN jar to
buffer B jar.
- Turn on lamp
In "Analysis" screen, set "Lamp" to "1".
Press "zero" button on SPD-10A.
- On computer:
Open "CLASS VP Chromatography".
Open "Corn HPLC".
the program is already opened in the computer.
- 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
- 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.
- In "sequence" screen (press
0 in main menu)
Set 'sample #' = 1 - 1
Set volume slightly less than sample volume, for example, 1 uL less
Set 'file' = F0
- Put sample vial in position
"1" on the sample rack.
- Press "single" on the
computer, enter directory and file name
- On system controller (SCL-10A),
press "run" button
- Wait for ~20 min for the DNA peak to
show up on the computer screen.
- 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.
- 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.
1. Turn off
lamp: set "Lamp" to 0 in "analysis" screen.
2. Turn off
In "analysis" screen, press "PMP.OFF" button (f1).
Wait until pump pressure drop to zero, then transfer pump B to 100% ACN.
Turn pump on again, until pressure equilibrates (600-700 psi for ACN).
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).