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protocol_468.ann
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T1 Action 29 35 Infect
E1 Action:T1 Acts-on:T45 Using:T47
T2 Action 100 108 Incubate
E2 Action:T2 Acts-on:T48 Using:T51 Using2:T3
T4 Action 181 191 Centrifuge
E4 Action:T4 Acts-on:T53 Site:T54
T5 Action 277 281 Save
E5 Action:T5 Acts-on:T58
T6 Action 300 304 Save
E6 Action:T6 Acts-on:T60
T7 Action 340 343 Add
E7 Action:T7 Acts-on:T63
T8 Action 431 441 Centrifuge
E8 Action:T8 Acts-on:T69 Using:T70
T9 Action 525 532 Discard
E9 Action:T9 Acts-on:T75
T10 Action 551 560 Resuspend
E10 Action:T10 Acts-on:T76 Using:T79 Using2:T82
T11 Action 631 639 Transfer
E11 Action:T11 Acts-on:T85 Site:T87
T12 Action 712 718 adjust
E12 Action:T12 Using:T90 Using2:T93
T13 Action 788 791 Add
E13 Action:T13 Acts-on:T95
T14 Action 814 817 mix
E14 Action:T14
T15 Action 819 827 Incubate
E15 Action:T15
T16 Action 860 863 Add
E16 Action:T16 Acts-on:T100 Site:T102
T17 Action 913 916 mix
E17 Action:T17
T18 Action 918 921 Add
E18 Action:T18 Acts-on:T104 Acts-on2:T107
T19 Action 981 984 mix
E19 Action:T19
T20 Action 986 994 Incubate
E20 Action:T20 Acts-on:T108
T21 Action 1030 1033 Add
E21 Action:T21 Acts-on:T113 Acts-on2:T115 Site:T117
T22 Action 1125 1128 Mix
E22 Action:T22 Using:T118
T23 Action 1143 1153 Centrifuge
E23 Action:T23 Using:T119
T24 Action 1206 1212 Remove
E24 Action:T24 Acts-on:T123 Site:T125
T25 Action 1254 1257 Add
E25 Action:T25 Acts-on:T127 Site:T129
T26 Action 1311 1314 Mix
E26 Action:T26 Using:T130
T27 Action 1332 1342 centrifuge
E27 Action:T27 Using:T133
T28 Action 1378 1384 Remove
E28 Action:T28 Acts-on:T134 Site:T136
T29 Action 1477 1482 Place
E29 Action:T29 Acts-on:T140 Site:T142
T30 Action 1532 1535 Add
E30 Action:T30 Acts-on:T145 Site:T147
T31 Action 1569 1580 Precipitate
E31 Action:T31 Acts-on:T148 Using:T153
T32 Action 1644 1647 Mix
E32 Action:T32
T33 Action 1657 1661 hold
E33 Action:T33
T34 Action 1682 1692 Centrifuge
E34 Action:T34 Acts-on:T157 Site:T158
T35 Action 1761 1768 Discard
E35 Action:T35 Acts-on:T163
T36 Action 1787 1791 Wash
E36 Action:T36 Acts-on:T164 Using:T168 Site:T169
T37 Action 1871 1874 Dry
E37 Action:T37 Acts-on:T172 Using:T175
T38 Action 1960 1966 remove
E38 Action:T38 Acts-on:T178 Using:E37
T39 Action 1977 1986 Resuspend
E39 Action:T39 Acts-on:T179 Using:T183
T40 Action 2085 2095 centrifuge
E40 Action:T40 Using:T184
T41 Action 2135 2141 remove
E41 Action:T41 Acts-on:T187 Site:T189
T42 Action 2175 2182 Discard
E42 Action:T42 Acts-on:T190
T43 Action 2196 2201 Store
E43 Action:T43 Acts-on:T191
T44 Amount 36 41 60 mL
T45 Reagent 45 54 chlorella
T46 Amount 60 66 200 µL
T47 Reagent 70 98 viral single plaque isolates
R1 Measure Arg1:T47 Arg2:T46
R2 Measure Arg1:T45 Arg2:T44
T48 Reagent 113 120 samples
T49 Temperature 124 128 25°C
T50 Time 133 144 24-72 hours
T51 Device 151 167 continuous light
R3 Setting Arg1:E2 Arg2:T49
R4 Setting Arg1:E2 Arg2:T50
T3 Method 172 179 shaking
T52 Amount 192 197 30 mL
T53 Reagent 205 212 lysates
T54 Device 220 238 Sorvall SS34 rotor
T55 Speed 242 251 5,000 rpm
T56 Time 265 270 5 min
T57 Temperature 272 275 4°C
R5 Measure Arg1:T53 Arg2:T52
R6 Setting Arg1:E4 Arg2:T55
R7 Setting Arg1:E4 Arg2:T56
R8 Setting Arg1:E4 Arg2:T57
T58 Reagent 286 298 supernatants
T59 Modifier 309 315 unused
T60 Reagent 316 323 portion
T61 Reagent 331 338 lysates
R9 Meronym Arg1:T60 Arg2:T61
R10 Mod-Link Arg1:T60 Arg2:T59
T62 Generic-Measure 344 347 10%
T63 Reagent 348 353 NP-40
T64 Reagent 358 370 Triton X-100
T65 Action 379 385 lysate
E3 Action:T65 Acts-on:T66 Using:E7
T66 Reagent 386 398 supernatants
T67 Measure-Type 404 423 final concentration
T68 Generic-Measure 427 429 1%
R11 Measure Arg1:T63 Arg2:T62
R12 Or Arg1:T63 Arg2:T64
R13 Measure Arg1:T64 Arg2:T62
R14 Setting Arg1:E3 Arg2:T68
R15 Of-Type Arg1:T68 Arg2:T67
T69 Reagent 446 454 material
T70 Device 458 479 Beckman Ti50.2 rotors
T71 Speed 483 493 15,000 rpm
T72 Speed 495 509 ~27,000 rcfmax
T73 Time 512 518 75 min
T74 Temperature 520 523 4°C
R16 Setting Arg1:E8 Arg2:T71
R17 Or Arg1:T71 Arg2:T72
R18 Setting Arg1:E8 Arg2:T73
R19 Setting Arg1:E8 Arg2:T74
T75 Reagent 537 549 supernatants
T76 Reagent 565 578 virus pellets
T77 Amount 584 590 1.0 mL
T78 Concentration 594 599 50 mM
T79 Reagent 600 608 Tris-HCl
T80 pH 610 616 pH 7.5
T81 Concentration 618 623 10 mM
T82 Reagent 624 629 MgCl2
R20 Measure Arg1:T82 Arg2:T81
R21 Measure Arg1:T79 Arg2:T78
R22 Measure Arg1:T79 Arg2:T77
R23 Measure Arg1:T79 Arg2:T80
T83 Amount 640 646 350 µL
T84 Modifier 654 665 resuspended
T85 Reagent 666 671 virus
T86 Amount 675 681 1.5 mL
T87 Location 682 707 screw-cap microfuge tubes
T88 Amount 739 745 500 µL
T89 Concentration 751 756 50 mM
T90 Reagent 757 765 Tris-HCl
T91 pH 767 773 pH 7.5
T92 Concentration 775 780 10 mM
T93 Reagent 781 786 MgCl2
R24 Measure Arg1:T93 Arg2:T92
R25 Measure Arg1:T90 Arg2:T89
R26 Setting Arg1:E12 Arg2:T88
R27 Mod-Link Arg1:T85 Arg2:T84
R28 Measure Arg1:T85 Arg2:T83
R29 Measure Arg1:T87 Arg2:T86
T94 Amount 792 798 8.8 µL
T95 Reagent 802 809 DNAse I
R30 Measure Arg1:T95 Arg2:T94
T96 Temperature 831 847 room temperature
T97 Time 852 858 60 min
R31 Setting Arg1:E15 Arg2:T96
R32 Setting Arg1:E15 Arg2:T97
T98 Amount 864 870 6.0 µL
T99 Concentration 874 880 500 mM
T100 Reagent 881 885 EDTA
T101 pH 887 893 pH 8.0
T102 Reagent 901 908 samples
R33 Measure Arg1:T100 Arg2:T99
R34 Measure Arg1:T100 Arg2:T98
R35 Measure Arg1:T100 Arg2:T101
T103 Amount 922 929 56.6 µL
T104 Reagent 933 945 proteinase K
T105 Amount 950 957 29.0 µL
T106 Concentration 961 964 10%
T107 Reagent 965 976 Na sarcosyl
R36 Measure Arg1:T107 Arg2:T106
R37 Measure Arg1:T107 Arg2:T105
R38 Measure Arg1:T104 Arg2:T103
T108 Reagent 999 1006 samples
T109 Temperature 1010 1017 60-65°C
T110 Time 1022 1028 60 min
R39 Setting Arg1:E20 Arg2:T109
R40 Setting Arg1:E20 Arg2:T110
T111 Amount 1034 1040 300 µL
T112 Modifier 1044 1060 buffer-saturated
T113 Reagent 1061 1067 phenol
T114 Amount 1072 1078 300 µL
T115 Reagent 1082 1103 CHCl3:Isoamyl alcohol
T116 Generic-Measure 1105 1109 24:1
T117 Location 1118 1123 tubes
R41 Mod-Link Arg1:T113 Arg2:T112
R42 Measure Arg1:T113 Arg2:T111
R43 Measure Arg1:T115 Arg2:T114
R44 Measure Arg1:T115 Arg2:T116
T118 Method 1132 1141 inversion
T119 Device 1161 1170 microfuge
T120 Speed 1174 1187 maximum speed
T121 Time 1192 1197 5 min
T122 Temperature 1201 1204 4°C
R45 Setting Arg1:E23 Arg2:T120
R46 Setting Arg1:E23 Arg2:T121
R47 Setting Arg1:E23 Arg2:T122
T123 Reagent 1217 1237 upper aqueous layers
T125 Location 1247 1252 tubes
T126 Amount 1258 1264 600 µL
T127 Reagent 1268 1289 CHCl3:Isoamyl alcohol
T128 Generic-Measure 1291 1295 24:1
T129 Location 1304 1309 tubes
R48 Measure Arg1:T127 Arg2:T126
R49 Measure Arg1:T127 Arg2:T128
T130 Method 1318 1327 inversion
T131 Time 1347 1352 5 min
T132 Temperature 1356 1359 4°C
T133 Device 1367 1376 microfuge
R50 Setting Arg1:E27 Arg2:T131
R51 Setting Arg1:E27 Arg2:T132
T134 Reagent 1389 1409 upper aqueous layers
T136 Location 1419 1424 tubes
T137 Action 1429 1435 repeat
E46 Action:T137 Count:T139 Using:T138
T138 Method 1440 1472 CHCl3:Isoamyl alcohol extraction
T139 Numerical 1473 1475 1X
T140 Reagent 1487 1502 last extraction
T141 Amount 1508 1514 2.0 mL
T142 Location 1515 1530 microfuge tubes
R52 Measure Arg1:T142 Arg2:T141
T143 Amount 1536 1541 66 µL
T144 Concentration 1545 1548 3 M
T145 Reagent 1549 1554 NaOAc
T146 Modifier 1558 1562 each
T147 Location 1563 1567 tube
R53 Mod-Link Arg1:T147 Arg2:T146
R54 Measure Arg1:T145 Arg2:T144
R55 Measure Arg1:T145 Arg2:T143
T148 Reagent 1585 1589 DNAs
T149 Amount 1595 1605 2X volumes
T150 Modifier 1607 1620 approximately
T151 Amount 1621 1628 1350 µL
T152 Concentration 1633 1637 100%
T153 Reagent 1638 1642 EtOH
R56 Measure Arg1:T153 Arg2:T152
R57 Mod-Link Arg1:T151 Arg2:T150
R58 Or Arg1:T149 Arg2:T151
R59 Measure Arg1:T153 Arg2:T149
T154 Modifier 1648 1652 well
T155 Temperature 1665 1670 -20°C
T156 Time 1671 1680 overnight
R60 Mod-Link Arg1:E32 Arg2:T154
R61 Setting Arg1:E33 Arg2:T155
R62 Setting Arg1:E33 Arg2:T156
T157 Location 1697 1702 tubes
T158 Device 1710 1719 microfuge
T159 Time 1724 1733 10-15 min
T160 Temperature 1737 1740 4°C
T161 Action 1744 1750 pellet
E47 Action:T161 Acts-on:T162 Using:E34
T162 Reagent 1755 1759 DNAs
R63 Setting Arg1:E34 Arg2:T159
R64 Setting Arg1:E34 Arg2:T160
T163 Reagent 1773 1785 supernatants
T164 Reagent 1796 1807 DNA pellets
T165 Concentration 1808 1810 1X
R65 Measure Arg1:T164 Arg2:T165
T166 Amount 1816 1823 1000 µL
T167 Concentration 1827 1830 70%
T168 Reagent 1831 1835 EtOH
T169 Device 1843 1852 microfuge
T170 Time 1857 1862 5 min
T171 Temperature 1866 1869 4°C
R66 Measure Arg1:T168 Arg2:T167
R67 Measure Arg1:T168 Arg2:T166
R68 Setting Arg1:E36 Arg2:T170
R69 Setting Arg1:E36 Arg2:T171
T172 Reagent 1879 1886 pellets
T173 Modifier 1887 1894 briefly
T174 Time 1896 1905 10-15 min
R70 Setting Arg1:T173 Arg2:T174
T175 Device 1914 1931 vacuum desiccator
T176 Device 1939 1948 speed vac
T177 Time 1950 1955 5 min
T178 Reagent 1971 1975 EtOH
R71 Mod-Link Arg1:T175 Arg2:T173
R72 Setting Arg1:T176 Arg2:T177
R73 Or Arg1:T175 Arg2:T176
T179 Reagent 1991 1995 DNAs
T180 Modifier 2001 2014 approximately
T181 Amount 2015 2020 60 µL
T182 Concentration 2024 2026 1X
T183 Reagent 2027 2036 TE buffer
R74 Measure Arg1:T183 Arg2:T182
R75 Measure Arg1:T183 Arg2:T181
R76 Mod-Link Arg1:T183 Arg2:T180
T184 Device 2103 2112 microfuge
T185 Time 2117 2123 15 min
T186 Temperature 2127 2130 4°C
T187 Reagent 2146 2158 supernatants
T189 Location 2168 2173 tubes
T135 Modifier 1413 1418 clean
R77 Mod-Link Arg1:T136 Arg2:T135
T124 Modifier 1241 1246 clean
R78 Mod-Link Arg1:T125 Arg2:T124
T188 Modifier 2162 2167 clean
R79 Setting Arg1:E40 Arg2:T185
R80 Setting Arg1:E40 Arg2:T186
R81 Mod-Link Arg1:T189 Arg2:T188
T190 Reagent 2187 2194 pellets
T191 Reagent 2206 2210 DNAs
T192 Temperature 2214 2217 4°C
R82 Setting Arg1:E43 Arg2:T192