Berglund:Sizing protocol

Sizing column protocol

1	Prime Äkta (all 4 pumps) 2	Turn Äkta on (on button on left hand side) 3	Turn on computer Unicorn program 4	Watch side of Äkta, when it asks you to press ok to syncronize the pumps, press ok 5	Go to System Control (see bottom of the screen) 6	Go to Manual Run (see at top of screen) 7	Choose Pump 8	Go to Äkta, place filter attached to tubing labelled A1 into water 9	Place all of the tubing under the Äkta into a collection vessel 10	Go to the computer, at Pump choose PumpWashPurifier 11	Choose A1 12	Click Execute 13	Wait for the procedure to finish 14	Go to Äkta, place filter A1 into sizing column buffer 15	Go to the computer, at Pump choose PumpWashPurifier 16	Click Execute 17	While running take 1ml green loop of tubing and connect one end to the 2 position and one end to the 6 position 18	Once procedure has finished, go to computer 19	Go to Alarms and Monitors 20	Click on Alarms_Pressure 21	Set pressure at 0.6MPa 22	Click Execute 23	Go to Flowpath 24	Choose ColumnPosition 25	Choose Column Position 3 (where the sizing column is) 26	Click Execute 27	Go to Pump 28	Click Flow 29	Set Flowrate at 0.2ml/min 30	Click Execute 31	Wait for 1 min, then raise Flowrate to 0.3ml/min 32	Click Execute 33	Wait for 1 min, then raise Flowrate to 0.4ml/min (the fastest possible) 34	Click Execute 35	Column is now ready to be used 36	Go to Flowpath 37	Choose InjectionValve 38	Choose Load 39	Click Execute 40	Get the 500µl syringe, suck up sizing buffer using the special loading needle 41	flick to get bubbles into the top of the syringe where the needle is 42	push bubbles out 43	place syringe needle into the metal loading piece between the two ends of the 44	green loop (injection port). Unscrew injection port, insert syringe, tighten port. 45	gently push buffer into the loop 46	LEAVE THE SYRINGE IN THE INJECTION PORT 47	Go to Flowpath 48	Choose InjectionValve 49	Choose Inject 50	Click Execute 51	wait 3 min. 52	Go to Flowpath 53	Choose InjectionValve 54	Choose Load 55	Click Execute 56	Repeat (from step 41) 57	Loop is now ready for sample to be injected. 58	Set waste tubes into a clean beaker to collect flow-through. 59	Try hard to not load more than 700µl of sample 60	Get a 1ml syringe, fill with sizing buffer using the loading needle 61	flick to get bubbles into the top of the syringe where the needle is 62	push bubbles out 63	push most of the buffer out, leave about 50µl 64	Suck up the sample to be loaded using the 1ml syringe 65	check to make sure there are no bubbles (very important) 66	place syringe needle into the injection port 67	gently push buffer into the loop 68	LEAVE THE SYRINGE IN THE INJECTION PORT 69	Go to Flowpath 70	Choose InjectionValve 71	Choose Inject 72	Click Execute 73	wait 10 min. 74	Go to Flowpath 75	Choose InjectionValve 76	Choose Load 77	Click Execute 78	Switch waste tubes back to waste 79	Go to Alarms and Monitors 80	Click on AutoZeroUV 81	Click Execute 82	VOID VOLUME IS 40ML FROM START OF LOAD (about 1.5hrs) 83	BEGIN COLLECTING AT 38ML 84	To collect fractions: 85	Replace all tubes in the fraction collector with fresh clean glass tubes 86	Center arm of collector over the first tube so that white paddle is directly center 87	on the glass tube 88	Go to Frac 89	Choose Fractionation_900 90	Choose to have 1ml samples collected 91	Click Execute 92	Go to Flowpath 93	Choose Outlet Valve 94	choose FracF2 95	Click Execute 96	collect fractions Once desired fractions have been collected it is time to clean up the Äkta and write down what was done in the log on the desk Go to Frac 97	Choose Fractionation_900 98	Choose to have 0ml samples collected 99	Click Execute 100	Go to Flowpath 101	Choose Outlet Valve 102	choose Waste F1 103	Click Execute 104	collect fractions 105	Go to Flowpath 106	Choose ColumnPosition 107	Choose Position 1bypass 108	Click Execute 109	Go to Alarms and Monitors 110	Click on Alarms_Pressure 111	Set pressure at 4MPa 112	Click Execute 113	Go to Pump 114	Click Flow 115	Set Flowrate at 4ml/min 116	Click Execute 117	Flush for 2 min. 118	Go to Pump 119	Click Flow 120	Set Flowrate at 0ml/min 121	Click Execute 122	Choose Pump 123	Go to Äkta, place filter attached to tubing labelled A1 into water 124	Go to the computer, at Pump choose PumpWashPurifier 125	Choose A1 126	Click Execute 127	Wait for the procedure to finish 128	Go to Äkta, place filter A1 into 20% Ethanol 129	Go to the computer, at Pump choose PumpWashPurifier 130	Click Execute 131	Write down pump pressure in the desk log 132	Wait for the procedure to finish 133	Click "End" 134	Exit the program, turn off the Äkta 135	You are done. :-)