protocol_132.ann

T1	Action 78 85	Aliquot
E1	Action:T1 Acts-on:T58 Site:T59
T2	Action 165 175	centrifuge
E2	Action:T2 
T3	Action 204 211	Discard
E3	Action:T3 Acts-on:T62
T4	Action 229 232	Add
E4	Action:T4 Acts-on:T63 Acts-on2:T64 Site:T65
T5	Action 292 298	vortex
E5	Action:T5 
T6	Action 313 316	Add
E6	Action:T6 Acts-on:T68
T7	Action 352 355	mix
E7	Action:T7 Using:E8
T8	Action 342 348	invert
E8	Action:T8 
T9	Action 361 368	ncubate
E9	Action:T9 Using:E10
T10	Action 406 412	mixing
E10	Action:T10 
T11	Action 414 422	Incubate
E11	Action:T11 Acts-on:T72
T13	Action 527 530	add
E13	Action:T13 Acts-on:T12 Acts-on2:T75
T14	Action 590 598	incubate
E14	Action:T14 Acts-on:T12
T15	Action 623 628	nvert
E15	Action:T15 Acts-on:T79
T16	Action 725 732	improve
E16	Action:T16 Measure-Type-Link:T85
T17	Action 677 683	digest
E17	Action:T17 Acts-on:T83
T18	Action 744 749	Allow
E18	Action:T18 Acts-on:T86
T19	Action 764 768	cool
E19	Action:T19 Acts-on:T86
T20	Action 790 793	Add
E20	Action:T20 Acts-on:T89
T21	Action 815 818	mix
E21	Action:T21 Acts-on:T90 Using:E22
T22	Action 822 831	inverting
E22	Action:T22 Count:T91
T23	Action 856 866	Centrifuge
E23	Action:T23 
T24	Action 908 914	remove
E24	Action:T24 Acts-on:T141
T25	Action 964 967	Add
E25	Action:T25 
T26	Action 1014 1020	invert
E26	Action:T26 
T27	Action 1043 1051	Incubate
E27	Action:T27 Acts-on:T145
T28	Action 1089 1092	Add
E28	Action:T28 Acts-on:T148
T29	Action 1126 1129	mix
E29	Action:T29 Using:E30
T30	Action 1133 1142	inverting
E30	Action:T30 Acts-on:T149 Count:T150
T31	Action 1167 1177	Centrifuge
E31	Action:T31 Acts-on:T151
T32	Action 1216 1224	Transfer
E32	Action:T32 Acts-on:T154
T33	Action 1261 1272	precipitate
E33	Action:T33 Acts-on:T157 Acts-on2:T158
T34	Action 1313 1319	Invert
E34	Action:T34 Acts-on:T159 Count:T160
T35	Action 1342 1353	precipitate
E35	Action:T35 Acts-on:T161
T37	Action 1443 1453	Centrifuge
E37	Action:T37 
T38	Action 1503 1509	Remove
E38	Action:T38 Acts-on:T132
T39	Action 1527 1530	Add
E39	Action:T39 Acts-on:T127 Site:T128
T40	Action 1565 1571	invert
E40	Action:T40 Count:T129
T41	Action 1610 1620	Centrifuge
E41	Action:T41 
T42	Action 1647 1653	Decant
E42	Action:T42 
T44	Action 1688 1694	Invert
E44	Action:T44 Acts-on:T115 Site:T118
T45	Action 1796 1799	Add
E45	Action:T45 Acts-on:T113 Site:T114
T47	Action 1830 1838	Incubate
E47	Action:T47 
T48	Action 1886 1895	rehydrate
E48	Action:T48 Using:E47
T49	Action 1960 1965	added
E49	Action:T49 Acts-on:T104
T50	Action 1981 1986	Store
E50	Action:T50 Acts-on:T102
T51	Action 2046 2056	Centrifuge
E51	Action:T51 
T53	Action 2120 2126	vortex
E53	Action:T53 Acts-on:T94
T54	Action 2184 2187	Add
E54	Action:T54 Acts-on:T57
T55	Action 2219 2222	mix
E55	Action:T55 Using:E56
T56	Action 2226 2235	inverting
E56	Action:T56 Acts-on:T92 Count:T93
T58	Reagent 86 132	0.5ml Gram positive bacteria overnight culture
T59	Location 140 160	1.5ml microfuge tube
T60	Speed 179 187	14,000xg
T61	Time 192 202	30 seconds
R1	Setting Arg1:E2 Arg2:T60	
R2	Setting Arg1:E2 Arg2:T61	
T62	Reagent 216 227	supernatant
T63	Reagent 233 252	450µl sterile water
T64	Reagent 257 266	50µl EDTA
T65	Location 274 280	pellet
T66	Modifier 285 291	gently
T67	Action 302 311	resuspend
E58	Action:T67 Using:E5
R3	Mod-Link Arg1:E5 Arg2:T66	
T68	Reagent 317 340	50µl Longlife™ Lysozyme
T69	Temperature 372 376	37°C
T70	Time 381 391	45 minutes
T71	Modifier 397 405	periodic
R4	Mod-Link Arg1:E10 Arg2:T71	
R5	Setting Arg1:E9 Arg2:T69	
R6	Setting Arg1:E9 Arg2:T70	
T72	Reagent 427 433	sample
T73	Temperature 437 444	55-60°C
T74	Time 449 459	15 minutes
R7	Setting Arg1:E11 Arg2:T73	
R8	Setting Arg1:E11 Arg2:T74	
T12	Reagent 531 556	1µl Proteinase K solution
T75	Reagent 567 585	100µl Lysis Buffer
T76	Modifier 561 566	every
T77	Time 611 620	1-2 hours
T78	Temperature 602 606	60°C
R9	Mod-Link Arg1:T75 Arg2:T76	
R10	Setting Arg1:E14 Arg2:T78	
R11	Setting Arg1:E14 Arg2:T77	
T79	Device 633 637	tube
T80	Modifier 638 650	periodically
T81	Time 651 660	each hour
R12	Mod-Link Arg1:T81 Arg2:T80	
R13	Setting Arg1:E15 Arg2:T81	
T82	Modifier 684 698	hard to handle
T83	Reagent 699 706	tissues
T84	Modifier 711 724	significantly
T85	Measure-Type 737 742	yield
R14	Mod-Link Arg1:T83 Arg2:T82	
R15	Mod-Link Arg1:E16 Arg2:T84	
T86	Reagent 754 760	sample
T87	Temperature 772 788	room temperature
R16	Setting Arg1:E19 Arg2:T87	
T88	Amount 794 799	200µl
T89	Reagent 800 810	chloroform
T90	Device 836 840	tube
T91	Numerical 841 848	several
R17	Measure Arg1:T89 Arg2:T88	
T57	Reagent 2188 2214	500µl Genomic Lysis Buffer
T92	Device 2240 2244	tube
T93	Numerical 2245 2252	several
T94	Device 2131 2135	tube
T95	Action 2139 2148	resuspend
E12	Action:T95 Acts-on:T96 Site:T97
T96	Reagent 2153 2159	pellet
T97	Reagent 2167 2182	residual liquid
T98	Modifier 2113 2119	Gently
R18	Mod-Link Arg1:E53 Arg2:T98	
T99	Time 2061 2070	5 minutes
T100	Speed 2074 2082	14,000xg
T101	Reagent 2100 2111	supernatant
T52	Action 2087 2095	pour off
E52	Action:T52 Acts-on:T101
R19	Setting Arg1:E51 Arg2:T99	
R20	Setting Arg1:E51 Arg2:T100	
T102	Reagent 1987 1990	DNA
T103	Temperature 1994 1997	4°C
R21	Setting Arg1:E50 Arg2:T103	
T104	Reagent 1907 1926	1µl LongLife™ RNase
T105	Modifier 1931 1936	every
T106	Reagent 1937 1952	100µl TE Buffer
R22	Mod-Link Arg1:T106 Arg2:T105	
T107	Action 2021 2026	store
E57	Action:T107 
T108	Temperature 2030 2035	-20°C
T109	Temperature 2039 2044	-80°C
R23	Setting Arg1:E57 Arg2:T108	
R24	Setting Arg1:E57 Arg2:T109	
T110	Temperature 1842 1858	room temperature
T111	Modifier 1863 1871	at least
T112	Time 1872 1882	15 minutes
R25	Setting Arg1:E47 Arg2:T110	
R26	Mod-Link Arg1:T112 Arg2:T111	
R27	Setting Arg1:E47 Arg2:T112	
T113	Reagent 1800 1814	50µl TE Buffer
T114	Location 1822 1828	pellet
T115	Device 1699 1703	tube
T116	Modifier 1709 1714	clean
T117	Modifier 1715 1724	absorbent
T118	Location 1725 1732	surface
T119	Time 1737 1752	several minutes
T120	Action 1756 1761	allow
E59	Action:T120 Acts-on:T122
T121	Modifier 1766 1772	excess
T122	Reagent 1773 1780	ethanol
T46	Action 1784 1794	drain away
E46	Action:T46 
R28	Mod-Link Arg1:T118 Arg2:T117	
R29	Mod-Link Arg1:T118 Arg2:T116	
R30	Setting Arg1:E44 Arg2:T119	
R31	Mod-Link Arg1:T122 Arg2:T121	
T123	Reagent 1673 1685	ethanol wash
T43	Action 1665 1668	off
E43	Action:T43 Using:E60 Using2:E42 Acts-on:T123
T124	Action 1657 1664	pipette
E60	Action:T124 
T125	Time 1625 1633	1 minute
T126	Speed 1637 1645	14,000xg
R32	Setting Arg1:E41 Arg2:T125	
R33	Setting Arg1:E41 Arg2:T126	
T128	Location 1556 1560	tube
T129	Numerical 1572 1579	several
T130	Action 1589 1593	wash
E61	Action:T130 Acts-on:T131 Using:E40
T131	Reagent 1598 1608	DNA pellet
T127	Reagent 1531 1548	700µl 70% ethanol
T132	Reagent 1514 1525	supernatant
T133	Speed 1457 1465	14,000xg
T134	Time 1470 1479	5 minutes
T135	Action 1483 1489	pellet
E62	Action:T135 Acts-on:T136
T136	Reagent 1490 1501	genomic DNA
R34	Setting Arg1:E37 Arg2:T133	
R35	Setting Arg1:E37 Arg2:T134	
T138	Time 871 881	10 minutes
T139	Speed 885 893	14,000xg
T140	Modifier 898 907	carefully
T141	Device 919 930	upper phase
T142	Action 936 941	clean
E63	Action:T142 Acts-on:T143
T143	Device 942 962	microcentrifuge tube
R36	Setting Arg1:E23 Arg2:T138	
R37	Setting Arg1:E23 Arg2:T139	
R38	Mod-Link Arg1:E24 Arg2:T140	
R39	Meronym Arg1:T141 Arg2:T143	
T144	Reagent 968 995	50µl DNA Stripping Solution
T145	Reagent 1056 1062	sample
T146	Time 1067 1079	5-10 minutes
T147	Temperature 1083 1087	60°C
R40	Setting Arg1:E27 Arg2:T146	
R41	Setting Arg1:E27 Arg2:T147	
T148	Reagent 1093 1121	100µl Precipitation Solution
T149	Device 1147 1151	tube
T150	Numerical 1152 1159	several
T151	Reagent 1182 1188	sample
T152	Speed 1192 1200	14,000xg
T153	Time 1205 1214	5 minutes
R42	Setting Arg1:E31 Arg2:T152	
R43	Setting Arg1:E31 Arg2:T153	
T154	Reagent 1229 1240	supernatant
T155	Modifier 1246 1251	clean
T156	Device 1252 1256	tube
R44	Mod-Link Arg1:T156 Arg2:T155	
T157	Reagent 1277 1288	genomic DNA
T158	Reagent 1294 1312	500µl isopropanol.
T159	Device 1324 1329	tubes
T160	Numerical 1330 1332	10
T161	Reagent 1358 1361	DNA