protocol_176.ann

T1	Action 77 84	Collect
E1	Action:T1 Acts-on:T46 Site:T47
T2	Action 167 174	isolate
E2	Action:T2 Acts-on:T45 Acts-on2:T46
T3	Action 197 205	Colonize
E3	Action:T3 Acts-on:T49
T4	Action 245 256	Anesthetize
E4	Action:T4 Acts-on:T50
T5	Action 352 357	split
E5	Action:T5 Acts-on:T58 Using:T57 Site:T59
T6	Action 412 421	pull back
E6	Action:T6 Acts-on:T61
T7	Action 466 471	Place
E7	Action:T7 Acts-on:T65
T8	Action 523 531	incubate
E8	Action:T8 Site:T70
T9	Action 772 779	Perform
E9	Action:T9 Using:T73 Using2:T74 Using3:T75 Site:T78 Using4:T77
T10	Action 894 898	Wash
E10	Action:T10 Acts-on:T80 Count:T81 Using:E48 Using2:T85
T11	Action 1000 1005	Treat
E11	Action:T11 Using:T88
T12	Action 1084 1096	Do not place
E12	Action:T12 Acts-on:T91
T13	Action 1125 1129	Stop
E13	Action:T13 Using:E49 Using2:E50
T14	Action 1226 1234	Post-fix
E14	Action:T14 Using:T102 Site2:T105 Using2:T100
T15	Action 1309 1313	Wash
E15	Action:T15 Acts-on:T106 Count:T107 Using:E51
T16	Action 1535 1541	Remove
E16	Action:T16 Acts-on:T112 Site:T113
T17	Action 1610 1618	Incubate
E17	Action:T17 Acts-on:T114 Site:T117
T18	Action 1681 1687	Change
E18	Action:T18 Acts-on:T120 Creates:T124
T19	Action 1736 1744	incubate
E19	Action:T19 
T20	Action 1771 1778	Prevent
E20	Action:T20 
T21	Action 1841 1846	place
E21	Action:T21 Acts-on:T128 Site:T129
T22	Action 1879 1883	seal
E22	Action:T22 Acts-on:T130 Using:T132
T24	Action 2740 2743	Mix
E24	Action:T24 Acts-on:T133 Site:T136
T25	Action 2914 2920	Remove
E25	Action:T25 Acts-on:T140
T26	Action 2956 2959	add
E26	Action:T26 Acts-on:T141 Site:T142
T27	Action 3184 3188	Wash
E27	Action:T27 Acts-on:T151 Site:T153
T28	Action 3252 3256	Wash
E28	Action:T28 Acts-on:T156 Site:T160 Site2:T161
T29	Action 3345 3349	Wash
E29	Action:T29 Acts-on:T165 Site:T168 Site2:T171
T30	Action 3438 3442	Wash
E30	Action:T30 Acts-on:T174 Site:T177 Site2:T180
T31	Action 3531 3535	Wash
E31	Action:T31 Acts-on:T183 Count:T184 Site:T186
T32	Action 3600 3604	Wash
E32	Action:T32 Acts-on:T187 Count:T188 Site:T190
T33	Action 3779 3787	Incubate
E33	Action:T33 Acts-on:T199 Site:T202
T34	Action 3864 3871	Aliquot
E34	Action:T34 Acts-on:T209 Site:T211
T35	Action 3972 3976	Heat
E35	Action:T35 Acts-on:T212 Using:T215
T36	Action 4060 4065	Store
E36	Action:T36 Acts-on:T217 Site:T218
T37	Action 4146 4153	Prepare
E37	Action:T37 Creates:T223 Using:E53
T38	Action 4361 4364	Mix
E38	Action:T38 Acts-on:T227 Site:T230
T39	Action 4661 4667	Remove
E39	Action:T39 Acts-on:T232 Site:T240
T40	Action 4791 4795	Wrap
E40	Action:T40 Acts-on:T241 Using:T242
T41	Action 4954 4958	Wash
E41	Action:T41 Acts-on:T244 Site:T247 Count:T245
T42	Action 5026 5030	Wash
E42	Action:T42 Acts-on:T250 Count:T251 Site:T253
T44	Action 362 371	peel back
E44	Action:T44 Acts-on:T58 Using:T57 Site:T59
T45	Location 180 195	collection cups
T46	Reagent 85 99	juvenile squid
T47	Location 105 119	hatching table
T48	Modifier 211 218	desired
T49	Reagent 219 237	V. fischeri strain
R1	Mod-Link Arg1:T49 Arg2:T48	
T50	Reagent 257 262	squid
T51	Time 267 272	2 min
T52	Action 276 283	placing
E45	Action:T52 Site:T54 Acts-on:T50
T53	Concentration 289 291	2%
T54	Reagent 292 299	ethanol
T55	Modifier 303 311	filtered
T56	Reagent 312 320	seawater
R2	Mod-Link Arg1:T56 Arg2:T55	
R3	Measure Arg1:T54 Arg2:T53	
R4	Setting Arg1:E4 Arg2:T51	
R5	Meronym Arg1:T54 Arg2:T56	
T57	Device 334 350	dissecting scope
T58	Reagent 372 378	mantle
T59	Location 382 395	anterior side
T60	Modifier 402 411	carefully
T61	Reagent 426 432	funnel
T62	Action 436 442	expose
E46	Action:T62 Acts-on:T63 Using:E6
T63	Location 447 458	light organ
R6	Mod-Link Arg1:E6 Arg2:T60	
T64	Modifier 472 481	dissected
T65	Reagent 482 487	squid
T66	Concentration 493 495	4%
T67	Reagent 496 512	paraformaldehyde
R7	Measure Arg1:T67 Arg2:T66	
R8	Mod-Link Arg1:T65 Arg2:T64	
T68	Time 532 541	overnight
T69	Temperature 545 549	4 °C
T70	Device 553 559	shaker
T71	Action 563 566	fix
E47	Action:T71 Using:E8
R9	Setting Arg1:T70 Arg2:T69	
R10	Setting Arg1:T70 Arg2:T68	
T72	Modifier 780 783	all
T73	Method 784 794	treatments
T74	Method 796 810	hybridizations
T75	Method 816 822	washes
T76	Amount 828 834	500-μL
T77	Device 848 862	rotator/shaker
T78	Location 835 841	volume
R11	Measure Arg1:T78 Arg2:T76	
T79	Modifier 899 903	each
T80	Location 904 910	sample
T81	Numerical 911 915	five
T82	Time 923 928	5 min
T83	Modifier 929 932	per
T84	Action 933 937	wash
E48	Action:T84 
R12	Mod-Link Arg1:E48 Arg2:T83	
R13	Setting Arg1:E48 Arg2:T82	
R14	Mod-Link Arg1:T80 Arg2:T79	
T85	Reagent 944 967	Permeabilization Buffer
T86	Temperature 971 987	room temperature
R15	Setting Arg1:E10 Arg2:T86	
T87	Concentration 1011 1021	0.01 mg/mL
T88	Reagent 1022 1058	Proteinase K Permeabilization Buffer
T89	Temperature 1062 1064	RT
T90	Time 1069 1083	15-20 minutes.
R16	Measure Arg1:T88 Arg2:T87	
R17	Setting Arg1:E11 Arg2:T89	
R18	Setting Arg1:E11 Arg2:T90	
T91	Device 1100 1106	shaker
T92	Device 1110 1117	rotator
R19	Or Arg1:T91 Arg2:T92	
T94	Reagent 1134 1146	proteinase K
T95	Action 1166 1172	washes
E50	Action:T95 Acts-on:T97 Site:T98 Count:T99
T96	Concentration 1176 1183	2 mg/mL
T97	Reagent 1184 1191	glycine
T98	Reagent 1195 1218	Permeabilization Buffer
R20	Measure Arg1:T97 Arg2:T96	
T99	Numerical 1162 1165	two
T93	Action 1147 1156	digestion
E49	Action:T93 Acts-on:T94
T100	Reagent 1238 1261	Permeabilization Buffer
T101	Concentration 1267 1269	4%
T102	Reagent 1270 1273	PFA
T103	Time 1278 1281	1 h
T104	Temperature 1285 1287	RT
R21	Measure Arg1:T102 Arg2:T101	
R22	Setting Arg1:E14 Arg2:T103	
R23	Setting Arg1:E14 Arg2:T104	
T105	Device 1295 1301	shaker
T106	Reagent 1319 1342	Permeabilization Buffer
T107	Numerical 1343 1347	five
T108	Time 1355 1360	5 min
T109	Modifier 1361 1364	per
T110	Action 1365 1369	wash
E51	Action:T110 
R24	Mod-Link Arg1:E51 Arg2:T109	
R25	Setting Arg1:E51 Arg2:T108	
T111	Modifier 1542 1549	as much
T112	Reagent 1557 1580	Permeabilization Buffer
T113	Reagent 1602 1608	sample
R26	Mod-Link Arg1:T112 Arg2:T111	
T114	Reagent 1619 1625	sample
T115	Amount 1629 1635	500 μL
T116	Concentration 1639 1642	50%
T117	Reagent 1643 1653	Hyb Buffer
T118	Temperature 1657 1662	65 °C
T119	Time 1667 1673	30 min
R27	Measure Arg1:T117 Arg2:T116	
R28	Measure Arg1:T117 Arg2:T115	
R29	Setting Arg1:E17 Arg2:T118	
R30	Setting Arg1:E17 Arg2:T119	
T120	Reagent 1688 1694	sample
T121	Amount 1700 1706	500 μL
T122	Modifier 1710 1715	fresh
T123	Concentration 1716 1719	50%
T124	Reagent 1720 1730	Hyb Buffer
T125	Temperature 1748 1753	65 °C
T126	Time 1758 1763	2.5 h
R31	Setting Arg1:E19 Arg2:T125	
R32	Setting Arg1:E19 Arg2:T126	
R33	Measure Arg1:T124 Arg2:T123	
R34	Mod-Link Arg1:T124 Arg2:T122	
R35	Measure Arg1:T124 Arg2:T121	
T127	Location 1827 1839	petri plates
T128	Mention 1847 1851	them
T129	Device 1857 1875	humidified chamber
R36	Coreference-Link Arg1:T128 Arg2:T127	
T130	Location 1888 1893	cover
T131	Location 1901 1906	plate
T132	Seal 1914 1931	strip of parafilm
R37	Meronym Arg1:T130 Arg2:T131	
T23	Generic-Measure 2744 2750	1 pmol
T43	Modifier 2754 2758	each
T133	Reagent 2759 2764	probe
T134	Amount 2768 2774	500 μL
T135	Concentration 2778 2781	50%
T136	Reagent 2782 2792	Hyb buffer
T137	Temperature 2796 2801	45 °C
T138	Time 2806 2812	30 min
R38	Mod-Link Arg1:T133 Arg2:T43	
R39	Measure Arg1:T133 Arg2:T23	
R40	Measure Arg1:T136 Arg2:T135	
R41	Measure Arg1:T136 Arg2:T134	
R42	Setting Arg1:E24 Arg2:T137	
R43	Setting Arg1:E24 Arg2:T138	
T139	Concentration 2925 2928	50%
T140	Reagent 2929 2939	Hyb buffer
T141	Reagent 2965 2979	probe solution
T142	Reagent 2983 2990	samples
T143	Time 2995 3004	overnight
T144	Action 3012 3022	incubation
E23	Action:T144 
T145	Temperature 3026 3032	45 °C.
R44	Measure Arg1:T140 Arg2:T139	
R45	Setting Arg1:E26 Arg2:T143	
R46	Setting Arg1:E23 Arg2:T145	
T146	Time 3006 3010	16 h
R47	Or Arg1:T143 Arg2:T146	
T147	Action 3079 3089	pre-warmed
E43	Action:T147 Acts-on:T149
T148	Temperature 3093 3098	45 °C
T149	Reagent 3051 3060	solutions
R48	Setting Arg1:E43 Arg2:T148	
T150	Modifier 3047 3050	All
R49	Mod-Link Arg1:T149 Arg2:T150	
T151	Reagent 3189 3196	samples
T152	Amount 3200 3206	500 μL
T153	Reagent 3207 3224	Probe Wash Buffer
T154	Time 3229 3235	15 min
T155	Temperature 3239 3244	45 °C
R50	Measure Arg1:T153 Arg2:T152	
R51	Setting Arg1:E27 Arg2:T154	
R52	Setting Arg1:E27 Arg2:T155	
T156	Reagent 3257 3264	samples
T157	Amount 3268 3274	500 μL
T158	Time 3322 3328	15 min
T159	Temperature 3332 3337	45 °C
T160	Reagent 3283 3300	Probe Wash Buffer
T161	Reagent 3313 3316	SSC
T162	Generic-Measure 3310 3312	5X
T163	Concentration 3303 3306	25%
R53	Measure Arg1:T161 Arg2:T162	
R54	Measure Arg1:T161 Arg2:T163	
T164	Concentration 3276 3279	75%
R55	Measure Arg1:T160 Arg2:T164	
R56	Measure Arg1:T160 Arg2:T157	
R57	Setting Arg1:E28 Arg2:T158	
R58	Setting Arg1:E28 Arg2:T159	
T165	Reagent 3350 3357	samples
T166	Amount 3361 3367	500 μL
T167	Concentration 3369 3372	50%
T168	Reagent 3376 3393	Probe Wash Buffer
T169	Concentration 3396 3399	50%
T171	Reagent 3403 3409	5X SSC
R61	Measure Arg1:T168 Arg2:T167	
T172	Time 3415 3421	15 min
T173	Temperature 3425 3430	45 °C
R62	Measure Arg1:T168 Arg2:T166	
R63	Setting Arg1:E29 Arg2:T172	
R64	Setting Arg1:E29 Arg2:T173	
T174	Reagent 3443 3450	samples
T175	Amount 3454 3460	500 μL
T176	Concentration 3462 3465	25%
T177	Reagent 3469 3486	Probe Wash Buffer
T178	Concentration 3489 3492	75%
T180	Reagent 3496 3502	5X SSC
R67	Measure Arg1:T177 Arg2:T176	
R68	Measure Arg1:T177 Arg2:T175	
T181	Time 3508 3514	15 min
T182	Temperature 3518 3523	45 °C
R69	Setting Arg1:E30 Arg2:T181	
R70	Setting Arg1:E30 Arg2:T182	
T183	Reagent 3536 3543	samples
T184	Numerical 3544 3545	2
T185	Amount 3556 3562	500 μL
T186	Reagent 3566 3572	5X SSC
R65	Measure Arg1:T180 Arg2:T178	
R59	Measure Arg1:T171 Arg2:T169	
R60	Measure Arg1:T171 Arg2:T166	
R66	Measure Arg1:T180 Arg2:T175	
R71	Measure Arg1:T186 Arg2:T185	
T170	Time 3577 3583	15 min
T179	Temperature 3587 3592	45 °C
R72	Setting Arg1:E31 Arg2:T170	
R73	Setting Arg1:E31 Arg2:T179	
T187	Reagent 3605 3612	samples
T188	Numerical 3613 3614	2
T189	Amount 3625 3631	500 μL
T190	Reagent 3635 3641	5X SSC
T191	Time 3646 3652	30 min
T192	Temperature 3656 3661	45 °C
R74	Measure Arg1:T190 Arg2:T189	
R75	Setting Arg1:E32 Arg2:T191	
R76	Setting Arg1:E32 Arg2:T192	
T193	Action 3700 3708	Incubate
E52	Action:T193 Acts-on:T194 Site:T196
T194	Reagent 3709 3716	samples
T195	Amount 3720 3726	500 μL
T196	Reagent 3730 3754	DNA Amplification Buffer
T197	Temperature 3758 3760	RT
T198	Time 3765 3771	30 min
R77	Measure Arg1:T196 Arg2:T195	
R78	Setting Arg1:E52 Arg2:T197	
R79	Setting Arg1:E52 Arg2:T198	
T199	Reagent 3788 3795	samples
T200	Modifier 3799 3804	fresh
T201	Amount 3805 3811	500 μL
T202	Reagent 3815 3839	DNA Amplification Buffer
T203	Temperature 3843 3845	RT
T204	Time 3850 3856	30 min
R80	Measure Arg1:T202 Arg2:T201	
R81	Mod-Link Arg1:T202 Arg2:T200	
R82	Setting Arg1:E33 Arg2:T203	
R83	Setting Arg1:E33 Arg2:T204	
T205	Amount 3872 3878	6 pmol
T206	Amount 3890 3896	100 μL
T207	Reagent 3900 3924	DNA Amplification Buffer
T208	Modifier 3929 3933	each
T209	Reagent 3934 3941	hairpin
T210	Modifier 3947 3955	separate
T211	Location 3956 3964	PCR tube
R84	Mod-Link Arg1:T211 Arg2:T210	
R85	Measure Arg1:T207 Arg2:T206	
R86	Mod-Link Arg1:T209 Arg2:T208	
R87	Measure Arg1:T209 Arg2:T205	
T212	Reagent 3981 3989	hairpins
T213	Temperature 3993 3998	95 °C
T214	Time 4003 4009	90 sec
R88	Setting Arg1:E35 Arg2:T213	
R89	Setting Arg1:E35 Arg2:T214	
T215	Device 4025 4051	PCR machine/thermal cycler
T216	Modifier 4070 4076	heated
T217	Reagent 4077 4085	hairpins
T218	Location 4093 4097	dark
T219	Time 4102 4108	30 min
T220	Temperature 4112 4114	RT
R90	Mod-Link Arg1:T217 Arg2:T216	
R91	Setting Arg1:E36 Arg2:T219	
R92	Setting Arg1:E36 Arg2:T220	
T221	Amount 4154 4160	100 μL
T222	Modifier 4164 4169	fresh
T223	Reagent 4170 4194	DNA Amplification Buffer
T224	Action 4195 4207	equilibrated
E53	Action:T224 
T225	Temperature 4211 4213	RT
R93	Setting Arg1:E53 Arg2:T225	
R94	Mod-Link Arg1:T223 Arg2:T222	
R95	Measure Arg1:T223 Arg2:T221	
T226	Modifier 4365 4368	all
T227	Reagent 4369 4377	hairpins
T228	Amount 4381 4387	100 μL
T229	Modifier 4391 4407	pre-equilibrated
T230	Reagent 4408 4432	DNA Amplification Buffer
T231	Temperature 4436 4438	RT
R96	Mod-Link Arg1:T227 Arg2:T226	
R97	Mod-Link Arg1:T230 Arg2:T229	
R98	Measure Arg1:T230 Arg2:T228	
R99	Setting Arg1:E38 Arg2:T231	
T232	Reagent 4668 4687	final wash solution
T234	Reagent 4711 4727	hairpin solution
T235	Reagent 4735 4742	samples
T236	Time 4750 4759	overnight
T237	Action 4767 4777	incubation
E54	Action:T237 
T238	Temperature 4781 4783	RT
R100	Setting Arg1:E54 Arg2:T238	
R101	Setting Arg1:E54 Arg2:T236	
T239	Time 4761 4765	16 h
R102	Or Arg1:T236 Arg2:T239	
T233	Action 4703 4706	add
E55	Action:T233 Acts-on:T234 Site:T235
T240	Location 4693 4698	[6.2]
T241	Location 4800 4812	sample tubes
T242	Seal 4837 4850	aluminum foil
T243	Action 4854 4868	keep light out
E56	Action:T243 Using:E40
T244	Reagent 4959 4966	samples
T245	Numerical 4967 4968	4
T246	Amount 4979 4985	500 μL
T247	Reagent 4989 4997	5X SSCTw
T248	Time 5002 5007	5 min
T249	Temperature 5016 5018	RT
T250	Reagent 5031 5038	samples
T251	Numerical 5039 5040	2
T252	Amount 5051 5057	500 μL
T253	Reagent 5061 5069	5X SSCTw
T254	Time 5074 5080	30 min
T255	Temperature 5089 5091	RT
R104	Measure Arg1:T247 Arg2:T246	
R105	Setting Arg1:E41 Arg2:T248	
R103	Setting Arg1:E41 Arg2:T249	
R106	Measure Arg1:T253 Arg2:T252	
R107	Setting Arg1:E42 Arg2:T254	
R108	Setting Arg1:E42 Arg2:T255	
T256	Action 5123 5129	imaged
E57	Action:T256 Acts-on:T259 Site:T258
T257	Modifier 5130 5138	directly
T258	Reagent 5142 5150	5X SSCTw
T259	Reagent 5108 5115	Samples
R109	Mod-Link Arg1:E57 Arg2:T257	
T260	Action 5155 5161	stored
E58	Action:T260 Acts-on:T261
T261	Reagent 5165 5173	5X SSCTw
T262	Temperature 5177 5181	4 °C
R110	Setting Arg1:E58 Arg2:T262