protocol_196.ann

T1	Action 50 54	Grow
E1	Action:T1 Acts-on:T48
T2	Action 79 90	Concentrate
E2	Action:T2 
T3	Action 112 115	Use
E3	Action:T3 Acts-on:T51
T4	Action 196 202	Infect
E4	Action:T4 Acts-on:T52
T5	Action 248 255	Harvest
E5	Action:T5 Acts-on:T53
T6	Action 315 322	Discard
E6	Action:T6 Acts-on:T57
T7	Action 339 351	flash freeze
E7	Action:T7 Acts-on:T58 Using:T59
T8	Action 379 384	Store
E8	Action:T8 
T9	Action 429 433	Take
E9	Action:T9 Acts-on:T62
T10	Action 491 496	break
E10	Action:T10 Using:T64 Acts-on:T63
T11	Action 529 532	add
E11	Action:T11 Acts-on:T66
T12	Action 623 628	Break
E12	Action:T12 Using:T68 Using2:T69 Acts-on:T67
T13	Action 668 673	Pipet
E13	Action:T13 Site:T71
T15	Action 810 815	Place
E15	Action:T15 
T16	Action 882 886	Thaw
E16	Action:T16 Acts-on:T81
T17	Action 925 928	Add
E17	Action:T17 Acts-on:T84
T18	Action 1011 1021	Centrifuge
E18	Action:T18 
T19	Action 1150 1153	Add
E19	Action:T19 Acts-on:T93
T20	Action 1206 1216	Centrifuge
E20	Action:T20 
T21	Action 1246 1253	Discard
E21	Action:T21 Acts-on:T99
T22	Action 1267 1271	Wash
E22	Action:T22 Acts-on:T100
T23	Action 1309 1319	Centrifuge
E23	Action:T23 
T24	Action 1345 1348	Dry
E24	Action:T24 Acts-on:T106 Site:T107
T25	Action 1475 1478	Add
E25	Action:T25 Acts-on:T113
T26	Action 1550 1558	Incubate
E26	Action:T26 
T27	Action 1573 1580	Extract
E27	Action:T27 Acts-on:T120
T28	Action 1630 1640	Centrifuge
E28	Action:T28 
T29	Action 1716 1719	Add
E29	Action:T29 Acts-on:T128
T30	Action 1768 1778	Centrifuge
E30	Action:T30 
T31	Action 1811 1818	Discard
E31	Action:T31 
T32	Action 1832 1836	Wash
E32	Action:T32 Acts-on:T135
T33	Action 1875 1885	Centrifuge
E33	Action:T33 
T34	Action 1911 1914	Dry
E34	Action:T34 Acts-on:T141 Using:T142
T35	Action 1977 1986	Resuspend
E35	Action:T35 Site:T145 Acts-on:T143
T36	Action 274 288	centrifugation
E36	Action:T36 
T37	Action 598 605	prevent
E37	Action:T37 
T38	Action 984 989	stand
E38	Action:T38 
T39	Action 1059 1067	Withdraw
E39	Action:T39 Acts-on:T90
T40	Action 1111 1118	deliver
E40	Action:T40 Site:T91
T41	Action 1178 1183	stand
E41	Action:T41 
T42	Action 1388 1398	DO NOT DRY
E42	Action:T42 
T43	Action 1432 1444	resuspending
E43	Action:T43 Acts-on:T109
T44	Action 1669 1677	Withdraw
E44	Action:T44 
T45	Action 1693 1696	add
E45	Action:T45 Acts-on:T125
T46	Action 1742 1747	stand
E46	Action:T46 
T47	Action 1954 1964	Do not dry
E47	Action:T47 
T48	Reagent 55 60	NC64A
T49	Concentration 94 110	1 x 108 cells/ml
R1	Setting Arg1:E2 Arg2:T49	
T50	Amount 116 121	30 ml
T51	Reagent 122 155	concentrated cells per time point
R2	Measure Arg1:T51 Arg2:T50	
T52	Reagent 208 213	virus
T53	Reagent 256 270	infected cells
T54	Speed 292 300	5000 rpm
T55	Time 302 307	5 min
T56	Temperature 310 313	4°C
R3	Setting Arg1:E36 Arg2:T54	
R4	Setting Arg1:E36 Arg2:T55	
R5	Setting Arg1:E36 Arg2:T56	
T57	Reagent 323 334	supernatant
T58	Reagent 352 358	pellet
T59	Reagent 362 377	liquid nitrogen
T60	Temperature 388 393	-80°C
R6	Setting Arg1:E8 Arg2:T60	
T61	Amount 434 440	1 to 2
T62	Reagent 441 448	samples
R7	Measure Arg1:T62 Arg2:T61	
T63	Reagent 497 504	pellets
T64	Device 516 523	spatula
T65	Amount 533 537	1 ml
T66	Reagent 538 552	TRIZOL reagent
R8	Measure Arg1:T66 Arg2:T65	
T67	Reagent 629 636	pellets
T68	Device 645 652	spatula
T69	Method 657 666	vortexing
T70	Modifier 679 683	2 ml
T71	Device 684 707	microcentrifuge tube(s)
R9	Mod-Link Arg1:T71 Arg2:T70	
T72	Amount 719 725	250 mg
T73	Modifier 729 753	0.25-0.30 mm baked glass
T74	Reagent 754 759	beads
R10	Mod-Link Arg1:T74 Arg2:T73	
R11	Measure Arg1:T74 Arg2:T72	
T14	Action 773 779	vortex
E14	Action:T14 
T75	Speed 783 796	highest speed
T76	Time 797 802	5 min
T77	Temperature 805 808	4°C
R12	Setting Arg1:E14 Arg2:T75	
R13	Setting Arg1:E14 Arg2:T76	
R14	Setting Arg1:E14 Arg2:T77	
T78	Temperature 819 824	-80°C
R15	Setting Arg1:E15 Arg2:T78	
T79	Time 825 834	overnight
R16	Setting Arg1:E15 Arg2:T79	
T80	Time 914 923	5 minutes
T81	Reagent 887 894	samples
T82	Action 899 905	vortex
E48	Action:T82 
R17	Setting Arg1:E48 Arg2:T80	
T83	Amount 929 935	250 µl
T84	Reagent 936 946	chloroform
R18	Measure Arg1:T84 Arg2:T83	
T85	Temperature 993 1002	room temp
T86	Time 1004 1009	3 min
R19	Setting Arg1:E38 Arg2:T85	
R20	Setting Arg1:E38 Arg2:T86	
T87	Speed 1033 1043	12,000 x g
T88	Time 1045 1051	15 min
T89	Temperature 1054 1057	4°C
R21	Setting Arg1:E18 Arg2:T87	
R22	Setting Arg1:E18 Arg2:T88	
R23	Setting Arg1:E18 Arg2:T89	
T90	Reagent 1072 1106	RNA-containing aqueous upper phase
T91	Device 1128 1148	microcentrifuge tube
T92	Amount 1154 1160	500 µl
T93	Reagent 1161 1172	isopropanol
R24	Measure Arg1:T93 Arg2:T92	
T94	Time 1184 1190	10 min
T95	Temperature 1195 1204	room temp
R25	Setting Arg1:E41 Arg2:T95	
R26	Setting Arg1:E41 Arg2:T94	
T96	Speed 1220 1230	12,000 x g
T97	Time 1232 1238	10 min
T98	Temperature 1241 1244	4°C
R27	Setting Arg1:E20 Arg2:T96	
R28	Setting Arg1:E20 Arg2:T97	
R29	Setting Arg1:E20 Arg2:T98	
T99	Reagent 1254 1265	supernatant
T100	Reagent 1272 1278	pellet
T101	Concentration 1284 1287	75%
T102	Reagent 1288 1292	EtOH
R30	Measure Arg1:T102 Arg2:T101	
T103	Speed 1320 1331	12,000 x g,
T104	Time 1332 1337	5 min
T105	Temperature 1340 1343	4°C
R31	Setting Arg1:E23 Arg2:T103	
R32	Setting Arg1:E23 Arg2:T104	
R33	Setting Arg1:E23 Arg2:T105	
T106	Reagent 1349 1356	pellets
T107	Device 1360 1377	vacuum dessicator
T108	Time 1378 1386	5-10 min
T109	Reagent 1450 1455	pelle
T110	Amount 1460 1466	250 µl
T111	Reagent 1467 1473	dd-H20
R34	Measure Arg1:T111 Arg2:T110	
T112	Amount 1479 1485	250 µl
T113	Reagent 1489 1496	2X MOPS
T114	Concentration 1498 1502	20mM
T115	Reagent 1503 1508	MgCl2
R35	Measure Arg1:T115 Arg2:T114	
R36	Measure Arg1:T113 Arg2:T112	
T116	Reagent 1529 1547	RNAse-free DNAse I
T117	Amount 1519 1526	100-200
R37	Measure Arg1:T116 Arg2:T117	
T118	Time 1559 1564	1-2 h
T119	Location 1568 1571	ice
T120	Reagent 1602 1619	phenol:chloroform
T121	Amount 1621 1627	500 µl
R38	Measure Arg1:T120 Arg2:T121	
T122	Speed 1644 1654	12,000 x g
T123	Time 1656 1661	5 min
T124	Temperature 1664 1667	4°C
R39	Setting Arg1:E28 Arg2:T122	
R40	Setting Arg1:E28 Arg2:T123	
R41	Setting Arg1:E28 Arg2:T124	
T125	Reagent 1697 1702	NaOAc
T126	Concentration 1706 1711	0.3 M
R42	Measure Arg1:T125 Arg2:T126	
T127	Amount 1720 1724	1 ml
T128	Reagent 1725 1736	isopropanol
R43	Measure Arg1:T128 Arg2:T127	
T129	Time 1748 1754	10 min
T130	Temperature 1757 1766	room temp
R44	Setting Arg1:E46 Arg2:T129	
R45	Setting Arg1:E46 Arg2:T130	
T131	Speed 1785 1795	12,000 x g
T132	Time 1797 1803	10 min
T133	Temperature 1806 1809	4°C
T134	Reagent 1819 1830	supernatant
T135	Reagent 1837 1843	pellet
T136	Concentration 1849 1853	75 %
T137	Reagent 1854 1858	EtOH
R46	Measure Arg1:T137 Arg2:T136	
T138	Speed 1886 1896	12,000 x g
R47	Setting Arg1:E33 Arg2:T138	
T139	Time 1898 1903	5 min
T140	Temperature 1906 1909	4°C
R48	Setting Arg1:E33 Arg2:T139	
R49	Setting Arg1:E33 Arg2:T140	
T141	Reagent 1915 1922	pellets
T142	Device 1926 1943	vacuum dessicator
T143	Reagent 1987 1994	pellets
T144	Amount 1998 2006	40-50 µl
T145	Reagent 2007 2012	water
R50	Measure Arg1:T145 Arg2:T144