HPLC 12/06/13

DNA labeling

• 60 nmol of DNA in 100 uL .1M NaHCO3 (100 mg in 12 mL)
• .1 – .2 mg dye in 50 uL DMSO (50 uL of 1-2 mg / mL)
• React at 37C for 1-2 hrs (shake occassionally).
• 300 nmol in 100 uL (5x solution). dilute 1:2.5 for 50 uL 2x solution. add .2M NaHCO3 (200 mg in 12 mL)
• react 50 uL dye, 50 uL DNA, 50 uL .2 M NaHCO3 for 2 hrs at 37C. Started 12:20p.

HPLC

1. start purging buffers A and B with Argon.
   • Use both the valves on the canister and on the tubing to turn Argon on and off
   • Be sure to rinse valves with H2O before and after placing into buffers.
2. Start Unicorn Software, log in as Bryan
3. check that instruments report they are ready

wash

1. go to Manual > Pump > PumpWashPurifer. Set Inlet A1 and B1 to ON.
2. check tubes A1 and B1 are going in 20% EtOH. Then hit execute.
3. Let run 2- 10 min, software will report in logbook when pump wash is done.
4. Now Select Flow, FLow Rate 2 (ml/min), hit execute
5. go to gradient, set to 50% B,hit execute
6. if graph looks flat after 1 min, okay to proceed.
7. Select Flowpath. Select OutletValve, FracF2, Execute. Liquid should come out of sample dispenser.
8. Select Frac, AccumulatorWash, 5 strokes, Execute. (this takes 5+ min, watch the log output).
9. Select Flowpath, InjectionValve, set Postion to Inject. Hit Execute. Run till clean / flat-line.
10. Select Pause from top bar. Stop Argon purge of bufffers.
11. Keep an aliquot of ~15 mL Buffer A for syringe wash and dissolving sample.
12. Attach buffers to their appropriate inputs.
13. Select Continue.
14. Select Manual > Pump > PumpWasPurifer (A1 and B1 should be ON). Execute.
15. Select Pump, Flow, FLow Rate 1 Execute. Gradient 100% B.
16. Check Column = C8. Unscrew bottom cap (writing goes from top to bottom). Unscrew orange cable. Drip into a column for 20s, Screw in top and screw in bottom.
17. column pressure should go up to around 4 MPa. Wash Column for 5 min. (can see x-axis).
18. meanwhile check that nothing is leaking out of the column (connections were made properly).
19. Rinse syringe with buffer A (just pippette and squirt into waste).
20. Column wash. Gradient: 0% B, 2 Min, Execute. (selecting y axis can change units to %B, or column pressure etc).
21. Once B reaches 0% and the pressure stabilizes, Fill syringe with BUffer A. Load through injection portal. Rpt 3x.
22. select Alarms&Mon in menu, UV1 260, UV3 699 (closet to 750 available)
23. select End from top menu when everything equilibrates

Sample loading

1. dissolve sample in 40 uL buffer A.
2. Hit Run, select program RNA collect, accept default settings, Integrate Full Report, Next, set savepath (alistairS3cy7). Select Start.
3. Inject sample within the first minute. (wait for gradients to equilibrate).
4. switch outlet path not necessary.
5. Manual, Alarms&Mon, confirm / reset wavelengths to monitor.
6. Select Frac, Fractionation, FracSize (.1) can do .2 if you want to spin longer.
7. start at FirstTube. When sample starts coming out, hit Execute. (15-20 min for cy5, Unlabeled DNA comes out around 8 min).