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protocol_233.ann
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T1 Reagent 127 141 Whole seawater
T2 Reagent 207 229 0.22-filtered seawater
T3 Action 287 295 filtered
E1 Action:T3 Acts-on:T1 Using:T5
T4 Size 315 322 0.02 µm
T5 Reagent 323 344 aluminum oxide filter
R1 Or Arg1:T1 Arg2:T2
R2 Measure Arg1:T5 Arg2:T4
T6 Action 461 467 remove
E2 Action:T6 Acts-on:T7 Using:T9
T7 Reagent 476 491 residual sample
T8 Amount 468 475;492 503 as much as possible
R3 Measure Arg1:T7 Arg2:T8
T9 Reagent 447 457 pumped air
T10 Action 515 519 seal
E3 Action:T10 Acts-on:T11 Using:T12
T11 Location 524 549 filter inlets and outlets
T12 Seal 555 563 parafilm
T13 Action 565 570 Label
E4 Action:T13 Acts-on:T14
T14 Location 575 581 filter
T15 Action 583 595 Flash-freeze
E5 Action:T15 Acts-on:T16 Using:T17
T16 Location 600 606 filter
T17 Reagent 610 625 liquid nitrogen
T18 Action 627 632 Store
E6 Action:T18 Acts-on:T19
T19 Mention 633 635 it
T20 Temperature 639 644 –80°C
R4 Setting Arg1:E6 Arg2:T20
T21 Reagent 663 682 Total nucleic acids
T22 Action 687 696 extracted
E7 Action:T22 Site:T23 Acts-on:T21 Using:T24
T23 Location 706 728 aluminum oxide filters
T24 Device 737 785 Masterpure complete DNA and RNA Purification kit
T25 Action 816 822 Remove
E8 Action:T25 Acts-on:T26 Site:T27 Using:T28
T26 Reagent 823 840 contaminating DNA
T27 Reagent 846 862 RNA preparations
T28 Device 872 911 Turbo DNA-free kit (Applied Biosystems)
T29 Modifier 912 962 as described in the protocol provided with the kit
R5 Mod-Link Arg1:E8 Arg2:T29
T30 Reagent 969 982 cDNA reaction
T31 Amount 992 997 11 µL
T32 Concentration 1048 1054 0.2 mM
T33 Amount 1073 1079 100 ng
T34 Amount 1114 1119 13 µL
T35 Reagent 1001 1046 extracted Turbo DNA-free–treated RNA template
T36 Reagent 1063 1067 dNTP
T37 Reagent 1083 1092 N6 primer
R6 Measure Arg1:T30 Arg2:T34
R7 Measure Arg1:T35 Arg2:T31
R8 Measure Arg1:T36 Arg2:T32
T38 Modifier 1058 1062 each
T39 Modifier 964 968 Each
R9 Mod-Link Arg1:T30 Arg2:T39
R10 Mod-Link Arg1:T36 Arg2:T38
R11 Measure Arg1:T37 Arg2:T33
R12 Meronym Arg1:T35 Arg2:T30
R13 Meronym Arg1:T36 Arg2:T30
R14 Meronym Arg1:T37 Arg2:T30
T40 Action 1193 1200 heating
E9 Action:T40 Acts-on:T41
T41 Reagent 1205 1211 sample
T42 Temperature 1215 1219 65°C
T43 Time 1224 1229 5 min
T44 Action 1239 1246 cooling
E10 Action:T44 Acts-on:T45 Using:T46
T45 Mention 1247 1249 it
R15 Coreference-Link Arg1:T45 Arg2:T41
R16 Setting Arg1:E9 Arg2:T42
R17 Setting Arg1:E9 Arg2:T43
T46 Location 1250 1256 on ice
T47 Reagent 1278 1281 DTT
T48 Concentration 1283 1289 0.5 mM
T49 Action 1306 1311 added
E11 Action:T49 Acts-on:T47 Site:T50
R18 Measure Arg1:T47 Arg2:T48
T50 Reagent 1319 1327 reaction
T51 Reagent 1373 1395 RNase OUT (Invitrogen)
T52 Action 1399 1406 protect
E12 Action:T52 Acts-on:T53 Using:E11
T53 Reagent 1411 1417 sample
T54 Amount 1368 1372 40 U
R19 Measure Arg1:T51 Arg2:T54
T55 Reagent 1443 1467 complementary DNA strand
T56 Action 1471 1482 synthesized
E13 Action:T56 Acts-on:T55 Using:T58
T57 Amount 1488 1493 200 U
T58 Reagent 1494 1544 Superscript III (Invitrogen) Reverse Transcriptase
R20 Measure Arg1:T58 Arg2:T57
T59 Reagent 1593 1601 reaction
T60 Action 1605 1614 incubated
E14 Action:T60 Acts-on:T59
T61 Temperature 1628 1632 25°C
T62 Time 1637 1642 5 min
R21 Setting Arg1:E14 Arg2:T61
R22 Setting Arg1:E14 Arg2:T62
T63 Temperature 1792 1796 50°C
T64 Time 1870 1876 60 min
T65 Action 1759 1770;1779 1788 temperature increased
E15 Action:T65
R23 Setting Arg1:E15 Arg2:T63
R24 Setting Arg1:E15 Arg2:T64
T66 Action 1882 1890;1908 1918 activity terminated
E16 Action:T66 Acts-on:T67 Using:E17
T67 Reagent 1898 1904 enzyme
T68 Action 1922 1932 incubating
E17 Action:T68 Acts-on:T69
T69 Reagent 1937 1945 reaction
T70 Temperature 1949 1953 85°C
T71 Time 1958 1962 5 mi
R25 Setting Arg1:E17 Arg2:T70
R26 Setting Arg1:E17 Arg2:T71
T72 Action 1987 1990 add
E18 Action:T72 Acts-on:T74 Site:T75
T73 Amount 1991 2001 1 µL (2 U)
T74 Reagent 2005 2025 RNase H (Invitrogen)
R27 Measure Arg1:T74 Arg2:T73
T75 Reagent 2033 2041 reaction
T76 Action 2046 2054 incubate
E19 Action:T76
T77 Temperature 2058 2062 37°C
T78 Time 2067 2073 20 min
T79 Action 2077 2083 digest
E20 Action:T79 Acts-on:T80 Using:E19
T80 Reagent 2088 2100 RNA template
T81 Reagent 2110 2127 cDNA:RNA molecule
R28 Meronym Arg1:T80 Arg2:T81
R29 Setting Arg1:E19 Arg2:T77
R30 Setting Arg1:E19 Arg2:T78
T82 Action 2129 2132;2140 2149 PCR performed
E21 Action:T82 Using:T87 Using2:T83
T87 Reagent 2159 2214 RdRp, Mpl.sc1, Mpl.sc2, Mplsc3, and Mpl.cdh primer sets
T83 Reagent 2241 2245 cDNA
T84 Action 2246 2257 synthesized
E22 Action:T84 Creates:T83 Creates2:T87
T85 Modifier 2215 2232 listed in Table 1
R31 Mod-Link Arg1:T87 Arg2:T85
T86 Location 2292 2314 nuclease-free PCR tube
T88 Size 2285 2291 0.2 mL
T89 Action 2316 2319 add
E23 Action:T89 Acts-on:T90 Site:T86
R32 Measure Arg1:T86 Arg2:T88
T90 Reagent 2320 2339 reaction components
T91 Concentration 2375 2377 1×
T92 Reagent 2378 2397 Platinum Taq buffer
T93 Concentration 2399 2403 3 mM
T94 Reagent 2404 2409 MgCl2
T95 Concentration 2411 2417 0.2 mM
T96 Reagent 2426 2430 dNTP
T97 Modifier 2421 2425 each
T98 Concentration 2432 2436 1 µM
T99 Modifier 2440 2444 each
T100 Reagent 2445 2451 primer
T101 Amount 2457 2463 1 unit
T102 Reagent 2467 2507 Platinum Taq DNA polymerase (Invitrogen)
R33 Measure Arg1:T102 Arg2:T101
R34 Measure Arg1:T92 Arg2:T91
T103 Action 2343 2371 achieve final concentrations
E24 Action:T103 Acts-on:T92 Acts-on2:T94 Acts-on3:T96 Acts-on4:T100 Acts-on5:T102 Using:E23
R35 Measure Arg1:T94 Arg2:T93
R36 Mod-Link Arg1:T96 Arg2:T97
R37 Measure Arg1:T96 Arg2:T95
R38 Mod-Link Arg1:T100 Arg2:T99
R39 Measure Arg1:T100 Arg2:T98
T104 Action 2509 2517 Incubate
E25 Action:T104 Acts-on:T105
T105 Reagent 2522 2531 reactions
T106 Temperature 2579 2583 94°C
T107 Time 2588 2592 75 s
R40 Setting Arg1:E25 Arg2:T106
R41 Setting Arg1:E25 Arg2:T107
T108 Temperature 2732 2736 94°C
T109 Time 2741 2745 45 s
T110 Action 2716 2728 denaturation
E26 Action:T110 Acts-on:T105
R42 Setting Arg1:E26 Arg2:T108
R43 Setting Arg1:E26 Arg2:T109
T111 Temperature 2827 2831 72°C
T112 Time 2836 2840 45 s
T113 Action 2747 2756 annealing
E27 Action:T113 Acts-on:T105
T114 Temperature 2762 2789 primer-specific temperature
T115 Time 2804 2808 45 s
T116 Action 2814 2823 extension
E28 Action:T116 Acts-on:T105
R44 Setting Arg1:E27 Arg2:T114
R45 Setting Arg1:E27 Arg2:T115
R46 Setting Arg1:E28 Arg2:T111
R47 Setting Arg1:E28 Arg2:T112
T117 Action 2946 2952 purify
E29 Action:T117 Acts-on:T119 Using:T120
T118 Action 2957 2968 concentrate
E30 Action:T118 Acts-on:T119 Using:T120
T119 Reagent 2973 2986 PCR reactions
T120 Device 2994 3030 MinElute PCR cleanup column (Qiagen)
T121 Reagent 3065 3086 Purified PCR products
T122 Action 3091 3097 loaded
E31 Action:T122 Acts-on:T121 Site:T124
T123 Concentration 3105 3107 1%
T124 Reagent 3108 3119 agarose gel
R48 Measure Arg1:T124 Arg2:T123
T125 Reagent 3131 3162 1× SYBR safe stain (Invitrogen)
R49 Meronym Arg1:T125 Arg2:T124
T126 Concentration 3167 3171 0.5×
T127 Reagent 3172 3182 TBE buffer
R50 Measure Arg1:T127 Arg2:T126
R51 Meronym Arg1:T127 Arg2:T125
T128 Action 3248 3255 excised
E32 Action:T128 Acts-on:T131 Using:T130
T129 Action 3260 3268 purified
E33 Action:T129 Acts-on:T131 Using:T130
T130 Device 3276 3312 MinElute Gel Extraction kit (Qiagen)
T131 Reagent 3184 3196 Bands of DNA
T132 Size 3222 3242 approximately 500 bp
R52 Measure Arg1:T131 Arg2:T132
T133 Action 3372 3379 eluting
E34 Action:T133 Acts-on:T134 Using:T137
T134 Reagent 3380 3383 DNA
T135 Location 3393 3399 column
R53 Meronym Arg1:T134 Arg2:T135
T136 Amount 3421 3426 10 µL
T137 Reagent 3427 3445 nuclease-free wate
R54 Measure Arg1:T137 Arg2:T136
T138 Reagent 3520 3532 PCR products
T139 Action 3537 3545 polished
E35 Action:T139 Acts-on:T138
T140 Action 3550 3564 phosphorylated
E36 Action:T140 Acts-on:T138 Using:T141
T141 Device 3574 3611 PCRTerminator End Repair kit (Lucigen
T142 Time 3655 3661 15-min
T143 Action 3662 3672 incubation
E37 Action:T143 Acts-on:T145
T144 Temperature 3676 3692 room temperature
T145 Reagent 3697 3705 reaction
T146 Action 3709 3717 purified
E38 Action:T146 Acts-on:T145 Using:T148
T147 Action 3722 3734 concentrated
E39 Action:T147 Using:T148 Acts-on:T145
T148 Device 3742 3783 MinElute Reaction Cleanup column (Qiagen)
R55 Setting Arg1:E37 Arg2:T142
R56 Setting Arg1:E37 Arg2:T144
T149 Reagent 3789 3808 eluted PCR products
T150 Action 3826 3833 ligated
E40 Action:T150 Acts-on:T149 Creates:T151
T151 Reagent 3843 3872 pSMART-HCKan vector (Lucigen)
T152 Time 3962 3968 15 min
T153 Temperature 3972 3976 70°C
T154 Amount 3978 3982 2 µL
T155 Action 3947 3957 incubating
E41 Action:T155 Acts-on:T161
R57 Setting Arg1:E41 Arg2:T152
R58 Setting Arg1:E41 Arg2:T153
T156 Reagent 3990 4007 ligation reaction
R59 Measure Arg1:T156 Arg2:T154
T157 Action 4011 4022 transformed
E42 Action:T157 Creates:T158 Acts-on:T156 Using:T159
T158 Reagent 4028 4062 Ecloni 10G Supreme cells (Lucigen)
T159 Method 4067 4082 electroporation
T160 Action 3910 3921 terminating
E43 Action:T160 Acts-on:T161 Using:E41
T161 Reagent 3926 3943 ligation reaction
T162 Action 4148 4157 screening
E44 Action:T162 Acts-on:T164
T163 Numerical 4158 4166 10 to 20
T164 Reagent 4167 4175 colonies
R60 Measure Arg1:T164 Arg2:T163
T165 Method 4191 4208 PCR amplification
T166 Reagent 4218 4237 primers SL1 and SR2
T167 Action 4251 4269 assess the quality
E45 Action:T167 Acts-on:T168 Using:E46
T168 Reagent 4277 4284 library
T169 Action 4180 4187 inserts
E46 Action:T169 Using:T165 Using2:T166
T170 Reagent 4286 4305 Colony PCR products
T171 Action 4313 4323 visualized
E47 Action:T171 Acts-on:T170 Site:T172
T172 Reagent 4329 4332 gel
T173 Reagent 4341 4349 products
T174 Action 4376 4384 purified
E48 Action:T174 Acts-on:T173
T175 Action 4389 4398 sequenced
E49 Action:T175 Acts-on:T173
T176 Modifier 4350 4375 in the correct size range
R61 Mod-Link Arg1:T173 Arg2:T176