ISCO Fraction Collector Directions (via Lindahl Lab)

1. Make sure UV machine settings are: noise filter=0.5, peak separator=off, sensitivity=1-2 ( usually 2) determines height of peaks

2. Turn on machine by pressing the STANDBY/OPERATE button to prime the system with d2H2O in bucket next to machine at 50%B at 60% speed. a. Press the ADVANCE button, Acc button, grad button, and set %B to 50%. (Hit End when done) b. Press the ADVANCE button, Acc button, Spd button, and set Pump speed to 60%. c. Turn pump on by pressing the ADVANCE button, press Pump to toggle on and off. (will hear pump start/stop) d. Let the machine run for about 60min to flush out air bubbles. (make sure tubes labeled A and B are in the water)

3. Prime the system with 60% sucrose by placing tubes A and B in beaker with 500ml of 60% sucrose. a. Leave machine at 50%B and 60% speed. b. Let the machine run for about 60min to flush and prime system.

4. Warm up the UV detector for about 30min by turning on by flipping switch from STANDBY to OPERATE. (the indicator light turn from red to green when ready) a. Zero the baseline of the detector by pressing AUTO BASELINE button and pen should go to 50% on recorder paper, if it doesn’t adjust by using the RECORDER OFFSET button.

5. Reverse the pump by moving switch on the TRIS pump to R for reverse (counterclockwise direction) to reverse direction of flow until the flow cell (SW40 tube) is down 2-3mm from the top then stop the pump by pressing the ADVANCE button and then Pump (while hear pump start)

6. Remove the SW40 tube from the piercer by carefully lowering the tube using the screw(s) on the right.

7. Change pumping direction to forward and start pump, making sure piercer needle is filled with 60% sucrose. Lower the piercer needle.

8. Install my SW40 tube with gradient and install from the top first being CAREFUL NOT TO SQUEESE TUBE.

9. Raise the apparatus to the tube and pierce with needle, making sure that it sure you are able to see hole in the needle in the tube. (just push needle up as far as possible) 10. Set CHART SPEED to 20 on the UV/VIS detector

11. Manually start pump at 50% gradient B at 20% speed and chart recorder at speed 60 at the same time.

12. Pump until the 60% sucrose fills tube to the bottom line then change gradient to 100% B at 60% speed and pump UNTIL COLLECTION TUBE IS FILLED, then STOP pump. a. Know when collection tube close to being filled when needle spikes and you can see the flow reaching the arm of the collector. b. BE READY TO STOP the pump as soon as it hits the black ring of the collector dispenser

13. Start program 2 by checking first it says Pgm=2 on the display and hit the Run button. a. It will collect ~0.5ml fractions by pumping 100%B 60% sucrose at 40% speed (1ml/min) into 26 Eppendorf tubes (put in 30 to be safe), press end when finished

14. Flush the system by pumping manually at 60% speed for 13min, re-zero chart recorder (put back to 10), then reverse pump for 4min until tubing in UV detector is clear and 2-3 mm of tube is empty.

15. Take tube of piercer, check that line to piecer needle is filled and insert next tube and repeat steps 7-13 until done.

16. After collection is complete, purge entire system with distilled water, remove and clean piecer apparatus, reinstall and PURGE AGAIN with water (60min), and turn off equipment

Precipitate significant fractions by adding 325ul of fraction to 30ul 100% TCA, mix will and incubate on ice for 30min and spin at 13000 rpm for 15min at 4C

Aspirate supernatant with pipet tip, resuspend in 30ul 0.2N NaOH, do RNA prep