- 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.
- 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.
- Start Unicorn Software, log in as Bryan
- check that instruments report they are ready
- go to Manual > Pump > PumpWashPurifer. Set Inlet A1 and B1 to ON.
- check tubes A1 and B1 are going in 20% EtOH. Then hit execute.
- Let run 2- 10 min, software will report in logbook when pump wash is done.
- Now Select Flow, FLow Rate 2 (ml/min), hit execute
- go to gradient, set to 50% B,hit execute
- if graph looks flat after 1 min, okay to proceed.
- Select Flowpath. Select OutletValve, FracF2, Execute. Liquid should come out of sample dispenser.
- Select Frac, AccumulatorWash, 5 strokes, Execute. (this takes 5+ min, watch the log output).
- Select Flowpath, InjectionValve, set Postion to Inject. Hit Execute. Run till clean / flat-line.
- Select Pause from top bar. Stop Argon purge of bufffers.
- Keep an aliquot of ~15 mL Buffer A for syringe wash and dissolving sample.
- Attach buffers to their appropriate inputs.
- Select Continue.
- Select Manual > Pump > PumpWasPurifer (A1 and B1 should be ON). Execute.
- Select Pump, Flow, FLow Rate 1 Execute. Gradient 100% B.
- 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.
- column pressure should go up to around 4 MPa. Wash Column for 5 min. (can see x-axis).
- meanwhile check that nothing is leaking out of the column (connections were made properly).
- Rinse syringe with buffer A (just pippette and squirt into waste).
- Column wash. Gradient: 0% B, 2 Min, Execute. (selecting y axis can change units to %B, or column pressure etc).
- Once B reaches 0% and the pressure stabilizes, Fill syringe with BUffer A. Load through injection portal. Rpt 3x.
- select Alarms&Mon in menu, UV1 260, UV3 699 (closet to 750 available)
- select End from top menu when everything equilibrates
- dissolve sample in 40 uL buffer A.
- Hit Run, select program RNA collect, accept default settings, Integrate Full Report, Next, set savepath (alistairS3cy7). Select Start.
- Inject sample within the first minute. (wait for gradients to equilibrate).
- switch outlet path not necessary.
- Manual, Alarms&Mon, confirm / reset wavelengths to monitor.
- Select Frac, Fractionation, FracSize (.1) can do .2 if you want to spin longer.
- start at FirstTube. When sample starts coming out, hit Execute. (15-20 min for cy5, Unlabeled DNA comes out around 8 min).
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