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.  :-)