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protocol_35.ann
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T1 Action 51 55 Grow
E1 Action:T1 Acts-on:T2 Using:T4
T2 Reagent 56 76 DH5alpha ecoli cells
T3 Time 77 86 overnight
T4 Reagent 92 113 pSP64 poly(A) plasmid
R1 Setting Arg1:E1 Arg2:T3
T5 Action 212 218 Set up
E2 Action:T5 Creates:T6 Site:T9 Using:T11 Using2:T13
T6 Reagent 223 241 digestion reaction
T7 Amount 245 249 50ul
T8 Concentration 250 252 1x
T9 Reagent 253 276 EcoRI compatible buffer
T10 Amount 282 286 ~3ug
T11 Reagent 287 306 pSP64-NSERT-poly(A)
T12 Amount 311 314 5ul
T13 Reagent 315 320 EcoRI
T14 Action 322 330 Incubate
E3 Action:T14
T15 Time 331 338 2 hours
T16 Temperature 345 348 37C
R6 Setting Arg1:E3 Arg2:T15
R7 Setting Arg1:E3 Arg2:T16
T17 Action 387 391 Pour
E4 Action:T17 Acts-on:T19
T18 Concentration 394 396 1%
T19 Reagent 397 408 agarose gel
T20 Action 413 416 run
E5 Action:T20 Acts-on:T22 Using:T23
T21 Amount 417 423 ~100ng
T22 Reagent 427 456 digest and undigested plasmid
T23 Method 461 478 check for cutting
T24 Action 521 529 clean up
E6 Action:T24 Acts-on:T25 Using:E7 Using2:T26
T25 Reagent 530 539 reactions
T26 Reagent 546 562 PCR clean up kit
T27 Action 564 571 pooling
E7 Action:T27 Acts-on:T28
T28 Reagent 572 606 repeats of the same plasmid digest
T29 Action 622 630 Quantify
E8 Action:T29 Using:T30 Measure-Type-Link:T31
T30 Device 637 645 nanodrop
T31 Measure-Type 666 679 concentration
T32 Action 681 687 Set up
E9 Action:T32 Creates:T33
T33 Reagent 688 711 SP6 reaction master mix
T34 Action 932 936 Make
E10 Action:T34 Site:T37 Creates:T36
T35 Amount 950 956 6.25ul
T36 Reagent 960 981 linarized plasmid DNA
T37 Location 941 946 tubes
T38 Action 1011 1017 dilute
E11 Action:T38 Acts-on:T40 Creates:T43 Using:T42
T39 Amount 1018 1021 1ul
T40 Reagent 1029 1046 provided standard
T41 Amount 1052 1058 5.25ul
T42 Reagent 1062 1067 water
T43 Reagent 989 1009 kit positive control
T44 Action 1125 1128 Add
E12 Action:T44 Acts-on:T46 Site:T47
T45 Amount 1129 1136 13.75ul
T46 Reagent 1140 1163 SP6 reaction master mix
T47 Location 1172 1176 tube
T48 Reagent 1180 1194 linearized DNA
R14 Meronym Arg1:T47 Arg2:T48
T49 Action 1196 1204 Incubate
E13 Action:T49 Site:T52
T50 Time 1205 1210 1-2hr
T51 Temperature 1214 1220 30/37C
T52 Device 1221 1231 water bath
R15 Setting Arg1:E13 Arg2:T50
T53 Action 1233 1239 Remove
E14 Action:T53 Site:T55 Creates:T56
T54 Amount 1240 1243 2ul
T55 Location 1249 1258 PCR tubes
T56 Reagent 1263 1272 later gel
T57 Action 1287 1290 add
E15 Action:T57 Acts-on:T59
T58 Amount 1291 1294 1ul
T59 Reagent 1298 1318 RQ1 RNase-Free DNase
T60 Action 1320 1328 Incubate
E16 Action:T60
T61 Time 1329 1334 15min
T62 Temperature 1338 1341 37C
R19 Setting Arg1:E16 Arg2:T61
R20 Setting Arg1:E16 Arg2:T62
T63 Action 1361 1364 add
E17 Action:T63 Acts-on:T65 Site:T68
T64 Amount 1365 1369 40ul
T65 Reagent 1370 1385 Ampure XP beads
T66 Action 1390 1393 mix
E18 Action:T66 Using:T67
T67 Device 1404 1411 pipette
T68 Reagent 1346 1359 each reaction
T69 Action 1417 1420 sit
E19 Action:T69
T70 Temperature 1421 1423 RT
T71 Time 1428 1432 5min
R22 Setting Arg1:E19 Arg2:T70
R23 Setting Arg1:E19 Arg2:T71
T72 Action 1506 1509 Add
E20 Action:T72 Acts-on:T75 Site:T76
T73 Amount 1510 1516 ~500ul
T74 Concentration 1517 1520 80%
T75 Reagent 1521 1525 etOH
T76 Reagent 1531 1536 beads
T77 Action 1542 1545 sit
E21 Action:T77
T78 Time 1546 1557 ~30 seconds
R26 Setting Arg1:E21 Arg2:T78
T79 Action 1564 1572 aspirate
E22 Action:T79
T80 Action 1578 1581 Add
E23 Action:T80 Acts-on:T83 Site:T84
T81 Amount 1582 1588 ~500ul
T82 Concentration 1589 1592 80%
T83 Reagent 1593 1597 etOH
T84 Reagent 1603 1608 beads
T85 Action 1614 1617 sit
E24 Action:T85
T86 Time 1618 1629 ~30 seconds
T87 Action 1636 1644 aspirate
E25 Action:T87
R29 Setting Arg1:E24 Arg2:T86
T88 Action 1727 1730 dry
E26 Action:T88 Acts-on:T89 Site:T90 Using:T91
T89 Reagent 1721 1726 beads
T90 Location 1738 1742 rack
T91 Device 1750 1757 kimwipe
T92 Time 1779 1784 10min
T93 Temperature 1785 1787 RT
R30 Setting Arg1:E26 Arg2:T92
R31 Setting Arg1:E26 Arg2:T93
T94 Action 1789 1798 Resuspend
E27 Action:T94 Acts-on:T95 Site:T97
T95 Reagent 1799 1804 beads
T96 Amount 1808 1812 20ul
T97 Reagent 1813 1825 hyclone H_2O
T98 Action 1827 1830 Run
E28 Action:T98 Acts-on:T101 Using:T105
T99 Amount 1831 1835 ~3ul
T100 Concentration 1839 1841 1%
T101 Reagent 1842 1857 agarose TAE gel
T102 Time 1868 1873 20min
T103 Generic-Measure 1877 1881 100V
T105 Device 1891 1905 NEB 1kb ladder
R36 Setting Arg1:E28 Arg2:T102
R37 Setting Arg1:E28 Arg2:T103
T106 Action 1963 1971 Quantify
E29 Action:T106 Using:T107
T107 Device 1975 1980 qubit
R2 Measure Arg1:T13 Arg2:T12
R3 Measure Arg1:T11 Arg2:T10
R4 Measure Arg1:T9 Arg2:T8
R5 Measure Arg1:T9 Arg2:T7
R8 Measure Arg1:T19 Arg2:T18
R9 Measure Arg1:T22 Arg2:T21
R10 Measure Arg1:T36 Arg2:T35
R12 Measure Arg1:T40 Arg2:T39
R11 Measure Arg1:T42 Arg2:T41
R13 Measure Arg1:T46 Arg2:T45
R17 Measure Arg1:T56 Arg2:T54
R18 Measure Arg1:T59 Arg2:T58
R21 Measure Arg1:T65 Arg2:T64
R24 Measure Arg1:T75 Arg2:T74
R25 Measure Arg1:T75 Arg2:T73
R27 Measure Arg1:T83 Arg2:T82
R28 Measure Arg1:T83 Arg2:T81
R32 Measure Arg1:T97 Arg2:T96
R34 Measure Arg1:T101 Arg2:T100
R35 Measure Arg1:T101 Arg2:T99
T108 Reagent 125 151 inserted spike-in sequence
R38 Meronym Arg1:T108 Arg2:T4
T109 Modifier 937 940 new
R39 Mod-Link Arg1:T37 Arg2:T109
T110 Modifier 1167 1171 each
R40 Mod-Link Arg1:T47 Arg2:T110
R41 Setting Arg1:T52 Arg2:T51
T111 Modifier 1394 1398 well
R16 Mod-Link Arg1:E18 Arg2:T111