-
Notifications
You must be signed in to change notification settings - Fork 3
/
Copy pathprotocol_491.ann
291 lines (291 loc) · 8.83 KB
/
protocol_491.ann
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267
268
269
270
271
272
273
274
275
276
277
278
279
280
281
282
283
284
285
286
287
288
289
290
291
T1 Action 35 41 Infect
E1 Action:T1 Acts-on:T43 Using:T45
T2 Action 115 123 Incubate
E2 Action:T2 Acts-on:T47 Using:T3
T4 Action 196 206 Centrifuge
E4 Action:T4 Acts-on:T51 Site:T52
T5 Action 292 295 Add
E5 Action:T5 Acts-on:T57 Site:T60 Measure-Type-Link:T61
T6 Action 384 394 Centrifuge
E6 Action:T6 Acts-on:T62 Using:T63
T7 Action 480 489 Resuspend
E7 Action:T7 Acts-on:T67 Site:T70 Site2:T73
T8 Action 560 568 Transfer
E8 Action:T8 Acts-on:T76 Site:T78
T9 Action 638 641 Add
E9 Action:T9 Acts-on:T80
T10 Action 662 665 mix
E10 Action:T10
T11 Action 667 670 Add
E11 Action:T11 Acts-on:T83 Site:T85
T12 Action 720 723 mix
E12 Action:T12
T13 Action 725 728 Add
E13 Action:T13 Acts-on:T87 Acts-on2:T89 Site:T91
T14 Action 820 830 Centrifuge
E14 Action:T14 Using:T92
T15 Action 883 889 Remove
E15 Action:T15 Acts-on:T96 Site:T98
T16 Action 931 934 Add
E16 Action:T16 Acts-on:T100 Site:T101
T17 Action 988 994 Remove
E17 Action:T17 Acts-on:T103 Site:T105
T18 Action 1087 1090 Add
E18 Action:T18 Acts-on:T111 Site:T113
T19 Action 1124 1135 Precipitate
E19 Action:T19 Acts-on:T114 Using:T118
T20 Action 1199 1209 Centrifuge
E20 Action:T20 Acts-on:T120 Using:T121
T21 Action 1278 1282 Wash
E21 Action:T21 Acts-on:T126 Using:T130 Site:T131
T22 Action 1362 1371 Resuspend
E22 Action:T22 Acts-on:T134 Using:T138
T23 Action 1423 1428 Store
E23 Action:T23 Acts-on:T139
T24 Action 1446 1449 Add
E24 Action:T24 Acts-on:T142 Acts-on2:T145
T25 Action 1505 1508 mix
E25 Action:T25
T26 Action 1510 1513 Dry
E26 Action:T26 Acts-on:T146 Site:T149
T27 Action 1574 1580 remove
E27 Action:T27 Acts-on:T150 Using:E26
T28 Action 1591 1595 Save
E28 Action:T28 Acts-on:T151
T29 Action 1614 1621 Discard
E29 Action:T29 Acts-on:T152
T30 Action 1640 1648 Incubate
E30 Action:T30
T31 Action 1681 1689 Incubate
E31 Action:T31 Acts-on:T155
T32 Action 1725 1728 Mix
E32 Action:T32 Using:T158
T33 Action 1743 1753 Centrifuge
E33 Action:T33 Using:T161
T34 Action 1789 1792 Mix
E34 Action:T34 Using:T162
T35 Action 1807 1812 Place
E35 Action:T35 Acts-on:T163 Site:T165
T36 Action 1862 1865 Mix
E36 Action:T36
T37 Action 1875 1879 hold
E37 Action:T37
T38 Action 1900 1907 Discard
E38 Action:T38 Acts-on:T169
T39 Action 1973 1983 centrifuge
E39 Action:T39 Using:T170
T40 Action 2020 2026 Remove
E40 Action:T40 Acts-on:T173 Site:T175
T41 Action 2060 2067 Discard
E41 Action:T41 Acts-on:T176
T42 Modifier 42 53 appropriate
T43 Reagent 54 58 host
T44 Modifier 64 71 desired
T45 Reagent 72 91 Chlorovirus species
T46 Generic-Measure 95 113 moi of ~0.001-0.01
R1 Mod-Link Arg1:T43 Arg2:T42
R2 Mod-Link Arg1:T45 Arg2:T44
R3 Setting Arg1:E1 Arg2:T46
T47 Reagent 128 135 samples
T48 Temperature 139 143 25°C
T49 Time 148 159 24-72 hours
T3 Method 166 194 continuous light and shaking
R4 Setting Arg1:E2 Arg2:T48
R5 Setting Arg1:E2 Arg2:T49
T50 Amount 207 212 30 mL
T51 Reagent 220 227 lysates
T52 Device 235 253 Sorvall SS34 rotor
T53 Speed 257 266 5,000 rpm
T54 Time 280 285 5 min
T55 Temperature 287 290 4°C
R6 Measure Arg1:T51 Arg2:T50
R7 Setting Arg1:E4 Arg2:T53
R8 Setting Arg1:E4 Arg2:T54
R9 Setting Arg1:E4 Arg2:T55
T56 Concentration 296 299 10%
T57 Reagent 300 305 NP-40
T58 Reagent 310 322 Triton X-100
T60 Reagent 331 350 lysate supernatants
T59 Concentration 380 382 1%
T61 Measure-Type 357 376 final concentration
R10 Measure Arg1:T57 Arg2:T56
R11 Or Arg1:T57 Arg2:T58
R12 Setting Arg1:E5 Arg2:T59
T62 Reagent 399 407 material
T63 Device 413 434 Beckman Ti 50.2 rotor
T64 Speed 438 448 15,000 rpm
T65 Time 467 473 75 min
T66 Temperature 475 478 4°C
R13 Setting Arg1:E6 Arg2:T64
R14 Setting Arg1:E6 Arg2:T65
R15 Setting Arg1:E6 Arg2:T66
T67 Reagent 494 507 virus pellets
T68 Amount 513 519 1.0 mL
T69 Concentration 523 528 50 mM
T70 Reagent 529 537 Tris-HCl
T71 pH 539 545 pH 7.5
T72 Concentration 547 552 10 mM
T73 Reagent 553 558 MgCl2
R16 Measure Arg1:T73 Arg2:T72
R17 Measure Arg1:T70 Arg2:T69
R18 Measure Arg1:T70 Arg2:T71
R19 Measure Arg1:T70 Arg2:T68
T74 Amount 569 575 500 µL
T75 Modifier 583 594 resuspended
T76 Reagent 595 600 virus
T77 Amount 604 610 1.5 ml
T78 Location 611 636 screw-cap microfuge tubes
R20 Measure Arg1:T78 Arg2:T77
R21 Mod-Link Arg1:T76 Arg2:T75
R22 Measure Arg1:T76 Arg2:T74
T79 Amount 642 646 9 µL
T80 Reagent 650 657 DNAse I
R23 Measure Arg1:T80 Arg2:T79
T81 Amount 671 677 6.0 µL
T82 Concentration 681 687 500 mM
T83 Reagent 688 692 EDTA
T84 pH 694 700 pH 8.0
T85 Reagent 708 715 samples
R24 Measure Arg1:T83 Arg2:T82
R25 Measure Arg1:T83 Arg2:T81
R26 Measure Arg1:T83 Arg2:T84
T86 Amount 729 735 300 µL
T87 Reagent 739 762 buffer-saturated phenol
T88 Amount 767 773 300 µL
T89 Reagent 777 798 CHCl3:Isoamyl alcohol
T90 Generic-Measure 800 804 24:1
T91 Location 813 818 tubes
R27 Measure Arg1:T89 Arg2:T90
R28 Measure Arg1:T89 Arg2:T88
R29 Measure Arg1:T87 Arg2:T86
T92 Device 838 847 microfuge
T93 Speed 851 864 maximum speed
T94 Time 869 874 5 min
T95 Temperature 878 881 4°C
R30 Setting Arg1:E14 Arg2:T93
R31 Setting Arg1:E14 Arg2:T94
R32 Setting Arg1:E14 Arg2:T95
T96 Reagent 894 914 upper aqueous layers
T97 Modifier 918 923 clean
T98 Location 924 929 tubes
R33 Mod-Link Arg1:T98 Arg2:T97
T99 Amount 935 941 600 µL
T100 Reagent 945 966 CHCl3:Isoamyl alcohol
T101 Location 981 986 tubes
T102 Generic-Measure 968 972 24:1
R34 Measure Arg1:T100 Arg2:T102
R35 Measure Arg1:T100 Arg2:T99
T103 Reagent 999 1019 upper aqueous layers
T104 Modifier 1023 1028 clean
T105 Location 1029 1034 tubes
T106 Action 1039 1045 repeat
E3 Action:T106 Using:T107 Count:T108
T107 Method 1050 1082 CHCl3:Isoamyl alcohol extraction
T108 Numerical 1083 1085 1X
R36 Mod-Link Arg1:T105 Arg2:T104
T109 Amount 1091 1096 66 µL
T110 Concentration 1100 1103 3 M
T111 Reagent 1104 1109 NaOAc
T112 Modifier 1113 1117 each
T113 Location 1118 1122 tube
R37 Mod-Link Arg1:T113 Arg2:T112
R38 Measure Arg1:T111 Arg2:T110
R39 Measure Arg1:T111 Arg2:T109
T114 Reagent 1140 1144 DNAs
T115 Modifier 1150 1160 2X volumes
T116 Amount 1176 1183 1350 µL
T117 Concentration 1188 1192 100%
T118 Reagent 1193 1197 EtOH
R40 Measure Arg1:T118 Arg2:T117
T119 Modifier 1162 1175 approximately
R42 Mod-Link Arg1:T116 Arg2:T119
R41 Measure Arg1:T118 Arg2:T116
R43 Setting Arg1:T115 Arg2:T116
R44 Mod-Link Arg1:T118 Arg2:T115
T120 Location 1214 1219 tubes
T121 Device 1227 1236 microfuge
T122 Time 1241 1250 10-15 min
T123 Temperature 1254 1257 4°C
T124 Action 1261 1267 pellet
E42 Action:T124 Acts-on:T125 Using:E20
T125 Reagent 1272 1276 DNAs
R45 Setting Arg1:E20 Arg2:T122
R46 Setting Arg1:E20 Arg2:T123
T126 Reagent 1287 1298 DNA pellets
T127 Concentration 1299 1301 1X
T128 Amount 1307 1314 1000 µL
T129 Concentration 1318 1321 70%
T130 Reagent 1322 1326 EtOH
T131 Device 1334 1343 microfuge
T132 Time 1348 1353 5 min
T133 Temperature 1357 1360 4°C
R47 Measure Arg1:T130 Arg2:T129
R48 Measure Arg1:T130 Arg2:T128
R49 Measure Arg1:T126 Arg2:T127
R50 Setting Arg1:E21 Arg2:T132
R51 Setting Arg1:E21 Arg2:T133
T134 Reagent 1376 1380 DNAs
T135 Modifier 1386 1399 approximately
T136 Amount 1400 1405 60 µL
T137 Concentration 1409 1411 1X
T138 Reagent 1412 1421 TE buffer
R52 Measure Arg1:T138 Arg2:T137
R53 Measure Arg1:T138 Arg2:T136
R54 Mod-Link Arg1:T136 Arg2:T135
T139 Reagent 1433 1437 DNAs
T140 Temperature 1441 1444 4°C
R55 Setting Arg1:E23 Arg2:T140
T141 Amount 1450 1455 57 µL
T142 Reagent 1459 1471 proteinase K
T143 Amount 1476 1481 29 µL
T144 Concentration 1485 1488 10%
T145 Reagent 1489 1500 Na sarcosyl
R56 Measure Arg1:T145 Arg2:T144
R57 Measure Arg1:T145 Arg2:T143
R58 Measure Arg1:T142 Arg2:T141
T146 Reagent 1518 1525 pellets
T147 Modifier 1526 1533 briefly
T148 Time 1535 1544 10-15 min
T149 Device 1553 1570 vacuum desiccator
T150 Reagent 1585 1589 EtOH
R59 Setting Arg1:T147 Arg2:T148
R60 Mod-Link Arg1:T146 Arg2:T147
T151 Reagent 1600 1612 supernatants
T152 Reagent 1626 1638 supernatants
T153 Temperature 1652 1668 room temperature
T154 Time 1673 1680 60 min.
R61 Setting Arg1:E30 Arg2:T153
R62 Setting Arg1:E30 Arg2:T154
T155 Reagent 1694 1701 samples
T156 Temperature 1705 1712 60-65°C
T157 Time 1717 1723 60 min
R63 Setting Arg1:E31 Arg2:T156
R64 Setting Arg1:E31 Arg2:T157
T158 Method 1732 1741 inversion
T159 Time 1758 1763 5 min
T160 Temperature 1767 1770 4°C
T161 Device 1778 1787 microfuge
R65 Setting Arg1:E33 Arg2:T159
R66 Setting Arg1:E33 Arg2:T160
T162 Method 1796 1805 inversion
T163 Reagent 1817 1832 last extraction
T164 Amount 1838 1844 2.0 mL
T165 Location 1845 1860 microfuge tubes
R67 Measure Arg1:T165 Arg2:T164
T166 Modifier 1866 1870 well
T167 Temperature 1883 1888 -20°C
T168 Time 1889 1898 overnight
R68 Setting Arg1:E37 Arg2:T167
R69 Setting Arg1:E37 Arg2:T168
R70 Mod-Link Arg1:E36 Arg2:T166
T169 Reagent 1912 1924 supernatants
T170 Device 1991 2000 microfuge
T171 Time 2005 2011 15 min
T172 Temperature 2015 2018 4°C
R71 Setting Arg1:E39 Arg2:T171
R72 Setting Arg1:E39 Arg2:T172
T173 Reagent 2031 2043 supernatants
T174 Modifier 2047 2052 clean
T175 Location 2053 2058 tubes
R73 Mod-Link Arg1:T175 Arg2:T174
T176 Location 2072 2079 pellets