-
Notifications
You must be signed in to change notification settings - Fork 3
/
Copy pathprotocol_238.ann
865 lines (865 loc) · 26.5 KB
/
protocol_238.ann
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267
268
269
270
271
272
273
274
275
276
277
278
279
280
281
282
283
284
285
286
287
288
289
290
291
292
293
294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
319
320
321
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
466
467
468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
489
490
491
492
493
494
495
496
497
498
499
500
501
502
503
504
505
506
507
508
509
510
511
512
513
514
515
516
517
518
519
520
521
522
523
524
525
526
527
528
529
530
531
532
533
534
535
536
537
538
539
540
541
542
543
544
545
546
547
548
549
550
551
552
553
554
555
556
557
558
559
560
561
562
563
564
565
566
567
568
569
570
571
572
573
574
575
576
577
578
579
580
581
582
583
584
585
586
587
588
589
590
591
592
593
594
595
596
597
598
599
600
601
602
603
604
605
606
607
608
609
610
611
612
613
614
615
616
617
618
619
620
621
622
623
624
625
626
627
628
629
630
631
632
633
634
635
636
637
638
639
640
641
642
643
644
645
646
647
648
649
650
651
652
653
654
655
656
657
658
659
660
661
662
663
664
665
666
667
668
669
670
671
672
673
674
675
676
677
678
679
680
681
682
683
684
685
686
687
688
689
690
691
692
693
694
695
696
697
698
699
700
701
702
703
704
705
706
707
708
709
710
711
712
713
714
715
716
717
718
719
720
721
722
723
724
725
726
727
728
729
730
731
732
733
734
735
736
737
738
739
740
741
742
743
744
745
746
747
748
749
750
751
752
753
754
755
756
757
758
759
760
761
762
763
764
765
766
767
768
769
770
771
772
773
774
775
776
777
778
779
780
781
782
783
784
785
786
787
788
789
790
791
792
793
794
795
796
797
798
799
800
801
802
803
804
805
806
807
808
809
810
811
812
813
814
815
816
817
818
819
820
821
822
823
824
825
826
827
828
829
830
831
832
833
834
835
836
837
838
839
840
841
842
843
844
845
846
847
848
849
850
851
852
853
854
855
856
857
858
859
860
861
862
863
864
865
T1 Action 63 69 Vortex
E1 Action:T1 Acts-on:T4
T2 Action 74 90 micro centrifuge
E2 Action:T2 Acts-on:T4
T3 Modifier 91 95 each
T4 Reagent 96 105 component
T5 Reagent 130 138 material
T6 Action 143 153 not lodged
E3 Action:T6 Acts-on:T5 Site:T7 Using:E2 Using2:E1
T7 Seal 161 164 cap
T8 Location 172 176 side
T9 Location 184 188 tube
T10 Action 190 193 Add
E4 Action:T10 Acts-on:T13 Site:T15 Creates:T19
T11 Amount 194 199 18 mL
T12 Modifier 203 226 molecular biology-grade
T13 Reagent 227 232 water
T14 Modifier 236 240 each
T15 Location 241 247 bottle
T16 Reagent 251 265 Elution Buffer
T17 Concentration 267 270 10X
T18 Concentration 282 284 1X
T19 Reagent 285 299 Elution Buffer
T20 Action 301 306 Check
E5 Action:T20 Acts-on:T21
T21 Location 307 310 box
T22 Location 314 320 bottle
T24 Reagent 329 334 water
T25 Action 344 349 added
E7 Action:T25 Acts-on:T24 Using:E5
T26 Action 368 372 come
E8 Action:T26 Acts-on:T28
T27 Concentration 357 360 50%
T28 Reagent 361 364 PEG
T29 Temperature 376 392 room temperature
T30 Action 415 423 Incubate
E9 Action:T30 Acts-on:T34 Using:T33
T31 Temperature 427 431 22°C
T32 Time 436 443 2 hours
T33 Device 449 461 thermocycler
T34 Reagent 405 414 Inhibitor
T35 Action 539 547 incubate
E10 Action:T35 Acts-on:T38
T36 Temperature 551 555 16°C
T37 Time 556 565 overnight
T38 Reagent 480 506 2’ O-methylated small RNAs
T39 Action 567 570 Add
E11 Action:T39 Acts-on:T45 Site:T47
T40 Amount 571 576 10 µL
T41 Amount 641 645 6 μL
T42 Amount 707 712 40 μL
T43 Amount 736 741 60 μL
T44 Modifier 580 593 Nuclease-free
T45 Reagent 594 599 Water
T46 Modifier 603 607 each
T47 Reagent 608 614 sample
T48 Action 619 622 mix
E12 Action:T48 Using:T49
T49 Method 626 635 pipetting
T50 Action 680 683 mix
E13 Action:T50 Using:T52
T51 Modifier 684 688 well
T52 Method 692 701 pipetting
T53 Reagent 649 675 Adapter Depletion Solution
T54 Reagent 716 731 AMPure XP beads
T55 Reagent 745 755 ispropanol
T56 Action 760 763 mix
E14 Action:T56 Using:T58
T57 Modifier 764 768 well
T58 Method 772 781 pipetting
T59 Action 783 791 Incubate
E15 Action:T59
T60 Time 796 805 5 minutes
T61 Action 807 816 Magnetize
E16 Action:T61 Acts-on:T62
T62 Reagent 817 822 beads
T63 Reagent 829 837 solution
T64 Action 848 854 Remove
E17 Action:T64 Acts-on:T66
T65 Action 859 866 discard
E18 Action:T65 Acts-on:T66
T66 Reagent 867 878 supernatant
T67 Action 889 892 Add
E19 Action:T67 Acts-on:T75
T68 Amount 893 899 180 μL
T69 Amount 1017 1023 180 μL
T70 Action 1013 1016 Add
E20 Action:T70 Acts-on:T76
T71 Modifier 903 919 freshly prepared
T72 Modifier 1027 1043 freshly prepared
T73 Concentration 920 923 80%
T74 Concentration 1044 1047 80%
T75 Reagent 924 931 ethanol
T76 Reagent 1048 1055 ethanol
T77 Action 933 941 incubate
E21 Action:T77
T78 Action 1057 1065 incubate
E22 Action:T78
T79 Time 946 956 30 seconds
T80 Time 1070 1080 30 seconds
T81 Action 962 968 remove
E23 Action:T81 Using:T83
T82 Action 1086 1092 remove
E24 Action:T82 Using:T84
T83 Device 976 980 P200
T84 Device 1100 1104 P200
T85 Device 984 988 P300
T86 Device 1108 1112 P300
T87 Amount 996 1002 200 μL
T88 Amount 1120 1126 200 μL
T89 Action 1128 1136 Incubate
E25 Action:T89 Acts-on:T90
T90 Reagent 1137 1143 sample
T91 Time 1148 1157 3 minutes
T92 Location 1235 1241 bottom
T93 Location 1249 1253 well
T94 Action 1218 1227 collected
E26 Action:T94 Site:T92 Creates:T98
T95 Action 1177 1183 remove
E27 Action:T95 Acts-on:T98
T96 Modifier 1188 1196 residual
T97 Modifier 1184 1187 all
T98 Reagent 1197 1203 liquid
T99 Action 1255 1261 Remove
E28 Action:T99 Acts-on:T100 Site:T101
T100 Location 1262 1267 plate
T101 Device 1273 1287 magnetic stand
T102 Action 1292 1301 resuspend
E29 Action:T102 Acts-on:T103 Site:T105 Using:T106
T103 Reagent 1302 1313 bead pellet
T104 Amount 1317 1322 22 μL
T105 Reagent 1326 1344 Resuspenion Buffer
T106 Method 1348 1376 pipetting volume up and down
T107 Action 1411 1422 resuspended
E30 Action:T107 Acts-on:T109
T108 Modifier 1400 1410 completely
T109 Reagent 1390 1395 beads
T110 Action 1424 1432 Incubate
E31 Action:T110
T111 Time 1433 1442 2 minutes
T112 Action 1463 1466 add
E32 Action:T112 Acts-on:T114 Site:T117
T113 Amount 1467 1471 6 μL
T114 Reagent 1475 1501 Adapter Depletion Solution
T115 Modifier 1507 1510 new
T116 Modifier 1512 1517 empty
T117 Location 1518 1522 well
T118 Action 1524 1533 Magnetize
E33 Action:T118 Acts-on:T119
T119 Reagent 1534 1540 sample
T120 Reagent 1547 1555 solution
T121 Action 1564 1569 clear
E34 Action:T121 Acts-on:T120 Using:E33
T122 Action 1571 1579 Transfer
E35 Action:T122 Acts-on:T124 Site:T125
T123 Amount 1580 1585 20 µL
T124 Reagent 1589 1600 supernatant
T125 Location 1608 1612 well
T126 Amount 1624 1628 6 µL
T127 Reagent 1629 1655 Adapter Depletion Solution
T128 Action 1660 1663 mix
E36 Action:T128 Using:T130
T129 Modifier 1664 1668 well
T130 Device 1672 1679 pipette
T131 Action 1681 1684 Add
E37 Action:T131 Acts-on:T133
T132 Amount 1685 1690 40 µL
T133 Reagent 1694 1709 AMPure XP beads
T134 Action 1711 1714 Add
E38 Action:T134 Acts-on:T136
T135 Amount 1715 1720 60 µL
T136 Reagent 1724 1735 isopropanol
T137 Action 1740 1743 mix
E39 Action:T137 Using:T139
T138 Modifier 1744 1748 well
T139 Method 1752 1761 pipetting
T140 Action 1763 1771 Incubate
E40 Action:T140
T141 Time 1776 1785 5 minutes
T142 Action 1787 1796 Magnetize
E41 Action:T142 Acts-on:T143
T143 Reagent 1797 1803 sample
T144 Reagent 1810 1818 solution
T145 Action 1827 1832 clear
E42 Action:T145 Acts-on:T144 Using:E41
T146 Action 1834 1840 Remove
E43 Action:T146 Acts-on:T148
T147 Action 1845 1852 discard
E44 Action:T147 Acts-on:T148
T148 Reagent 1853 1864 supernatant
T149 Action 1875 1878 Add
E45 Action:T149 Acts-on:T157
T150 Action 1999 2002 Add
E46 Action:T150 Acts-on:T158
T151 Amount 1879 1885 180 µL
T152 Amount 2003 2009 180 µL
T153 Modifier 1889 1905 freshly prepared
T154 Modifier 2013 2029 freshly prepared
T155 Concentration 1906 1909 80%
T156 Concentration 2030 2033 80%
T157 Reagent 1910 1917 ethanol
T158 Reagent 2034 2041 ethanol
T159 Action 1919 1927 incubate
E47 Action:T159
T160 Action 2043 2051 incubate
E48 Action:T160
T161 Time 1932 1942 30 seconds
T162 Time 2056 2066 30 seconds
T163 Action 1948 1954 remove
E49 Action:T163 Acts-on:T165
T164 Action 2072 2078 remove
E50 Action:T164 Acts-on:T166
T165 Device 1962 1966 P200
T166 Device 2086 2090 P200
T167 Device 1970 1974 P300
T168 Device 2094 2098 P300
T169 Amount 1982 1988 200 µL
T170 Amount 2106 2112 200 µL
T171 Action 2114 2122 Incubate
E51 Action:T171 Acts-on:T172
T172 Reagent 2123 2129 sample
T173 Time 2134 2143 3 minutes
T174 Time 2151 2161 one minute
T175 Action 2163 2169 remove
E52 Action:T175 Acts-on:T178
T176 Modifier 2170 2173 all
T177 Modifier 2174 2182 residual
T178 Reagent 2183 2189 liquid
T179 Action 2203 2212 collected
E53 Action:T179 Site:T180 Creates:T178
T180 Location 2220 2226 bottom
T181 Location 2234 2238 well
T182 Action 2240 2246 Remove
E54 Action:T182 Acts-on:T183 Site:T184
T183 Location 2247 2252 plate
T184 Device 2258 2272 magnetic stand
T185 Action 2277 2286 resuspend
E55 Action:T185 Acts-on:T186 Site:T188 Using:T189
T186 Reagent 2287 2298 bead pellet
T187 Amount 2302 2307 12 µL
T188 Reagent 2311 2329 Nuclease-freeWater
T189 Method 2333 2361 pipetting volume up and down
T190 Action 2396 2407 resuspended
E56 Action:T190 Acts-on:T192
T191 Modifier 2385 2395 completely
T192 Reagent 2375 2380 beads
T193 Action 2409 2417 Incubate
E57 Action:T193
T194 Time 2422 2431 2 minutes
T195 Action 2433 2442 Magnetize
E58 Action:T195 Acts-on:T196
T196 Reagent 2443 2449 sample
T197 Reagent 2456 2464 solution
T198 Action 2473 2478 clear
E59 Action:T198 Acts-on:T197 Using:E58
T199 Action 2480 2488 Transfer
E60 Action:T199 Acts-on:T201 Site:T203
T200 Amount 2489 2494 11 µL
T201 Reagent 2498 2509 supernatant
T202 Modifier 2515 2518 new
T203 Location 2519 2523 well
T204 Action 2544 2547 add
E61 Action:T204 Acts-on:T206
T205 Amount 2548 2554 1.5 µL
T206 Reagent 2562 2585 5’ NEXTflex™ 4N Adapter
T207 Action 2590 2594 heat
E62 Action:T207
T208 Temperature 2598 2602 70°C
T209 Time 2607 2616 2 minutes
T210 Location 2644 2647 ice
T211 Action 2635 2640 place
E63 Action:T211 Site:T210
T212 Modifier 2623 2634 immediately
T213 Action 2649 2657 Incubate
E64 Action:T213 Using:T216
T214 Temperature 2661 2665 20°C
T215 Time 2670 2676 1 hour
T216 Device 2682 2694 thermocycler
T217 Action 2696 2699 Add
E65 Action:T217 Acts-on:T219 Site:T221
T218 Amount 2700 2704 1 µL
T219 Reagent 2705 2724 NEXTflex™ RT primer
T220 Modifier 2728 2732 each
T221 Reagent 2733 2739 sample
T222 Action 2741 2745 Heat
E66 Action:T222
T223 Temperature 2749 2753 70°C
T224 Time 2758 2767 2 minutes
T225 Modifier 2773 2784 immediately
T226 Action 2785 2790 place
E67 Action:T226 Site:T227
T227 Location 2794 2797 ice
T228 Reagent 2808 2814 sample
T229 Modifier 2803 2807 each
T230 Action 2816 2819 add
E68 Action:T230 Acts-on:T231 Site:T228
T231 Reagent 2824 2844 following components
T232 Action 2849 2852 mix
E69 Action:T232
T233 Modifier 2853 2857 well
T234 Action 2859 2867 Incubate
E70 Action:T234 Using:T235
T235 Device 2873 2885 thermocycler
T236 Temperature 2889 2893 44°C
T237 Time 2898 2904 1 hour
T238 Temperature 2917 2921 90°C
T239 Time 2926 2936 10 minutes
T240 Reagent 2991 2998 samples
T241 Action 2999 3005 stored
E71 Action:T241 Acts-on:T240
T242 Temperature 3009 3014 -20°C
T243 Action 2966 2973 stopped
E72 Action:T243
T244 Method 2977 2986 this step
T245 Modifier 2959 2965 safely
T246 Method 2942 2951 procedure
T247 Action 3016 3019 Add
E73 Action:T247 Acts-on:T251 Site:T255
T248 Action 3098 3101 Add
E74 Action:T248 Acts-on:T252 Acts-on2:T254
T249 Amount 3020 3025 10 µL
T250 Amount 3102 3107 40 µL
T251 Reagent 3029 3055 Adapter Depletion Solution
T252 Reagent 3111 3126 AMPure XP beads
T253 Amount 3131 3136 90 µL
T254 Reagent 3140 3150 ispropanol
T255 Reagent 3064 3070 sample
T256 Modifier 3059 3063 each
T257 Action 3075 3078 mix
E75 Action:T257 Using:T261
T258 Action 3155 3158 mix
E76 Action:T258 Using:T262
T259 Modifier 3079 3083 well
T260 Modifier 3159 3163 well
T261 Device 3087 3094 pipette
T262 Device 3167 3174 pipette
T263 Action 3176 3184 Incubate
E77 Action:T263
T264 Time 3189 3198 5 minutes
T265 Action 3200 3209 Magnetize
E78 Action:T265 Acts-on:T266
T266 Reagent 3210 3216 sample
T267 Reagent 3223 3231 solution
T268 Action 3235 3240 clear
E79 Action:T268 Acts-on:T267 Using:E78
T269 Action 3242 3248 Remove
E80 Action:T269 Acts-on:T271
T270 Action 3253 3260 discard
E81 Action:T270 Acts-on:T271
T271 Reagent 3261 3272 supernatant
T272 Action 3283 3286 Add
E82 Action:T272 Acts-on:T280
T273 Action 3407 3410 Add
E83 Action:T273 Acts-on:T281
T274 Amount 3287 3293 180 µL
T275 Amount 3411 3417 180 µL
T276 Modifier 3297 3313 freshly prepared
T277 Modifier 3421 3437 freshly prepared
T278 Concentration 3314 3317 80%
T279 Concentration 3438 3441 80%
T280 Reagent 3318 3325 ethanol
T281 Reagent 3442 3449 ethanol
T282 Action 3327 3335 incubate
E84 Action:T282
T283 Action 3451 3459 incubate
E85 Action:T283
T284 Time 3340 3350 30 seconds
T285 Time 3464 3474 30 seconds
T286 Action 3356 3362 remove
E86 Action:T286 Acts-on:T288
T287 Action 3480 3486 remove
E87 Action:T287 Acts-on:T289
T288 Device 3370 3374 P200
T289 Device 3494 3498 P200
T290 Device 3378 3382 P300
T291 Device 3502 3506 P300
T292 Amount 3390 3396 200 µL
T293 Amount 3514 3520 200 µL
T294 Action 3522 3530 Incubate
E88 Action:T294 Acts-on:T295
T295 Reagent 3531 3537 sample
T296 Time 3542 3551 3 minutes
T297 Action 3571 3577 remove
E89 Action:T297 Acts-on:T300
T298 Modifier 3578 3581 any
T299 Modifier 3582 3590 residual
T300 Reagent 3591 3597 liquid
T301 Location 3628 3634 bottom
T302 Location 3642 3646 well
T303 Time 3559 3569 one minute
T304 Action 3648 3654 Remove
E90 Action:T304 Acts-on:T305 Site:T306
T305 Location 3655 3660 plate
T306 Device 3666 3680 magnetic stand
T307 Action 3685 3694 resuspend
E91 Action:T307 Acts-on:T308 Site:T310 Using:T311
T308 Reagent 3695 3706 bead pellet
T309 Amount 3710 3715 19 µL
T310 Reagent 3719 3737 Nuclease-freeWater
T311 Method 3741 3750 pipetting
T313 Reagent 3764 3769 beads
T314 Modifier 3774 3784 completely
T315 Action 3785 3796 resuspended
E93 Action:T315 Acts-on:T313
T316 Action 3798 3806 Incubate
E94 Action:T316
T317 Time 3811 3820 2 minutes
T318 Action 3822 3831 Magnetize
E95 Action:T318
T319 Reagent 3838 3846 solution
T320 Action 3850 3855 clear
E96 Action:T320 Acts-on:T319
T321 Action 3857 3865 Transfer
E97 Action:T321 Acts-on:T323 Site:T325
T322 Amount 3866 3871 18 µL
T323 Reagent 3875 3886 supernatant
T324 Modifier 3892 3895 new
T325 Location 3896 3900 well
T326 Action 3902 3905 Add
E98 Action:T326 Acts-on:T329 Site:T331
T327 Amount 3906 3910 5 µL
T328 Concentration 3914 3916 6X
T329 Reagent 3917 3932 Gel Loading Dye
T330 Modifier 3936 3940 each
T331 Reagent 3941 3952 PCR product
T332 Action 3957 3960 mix
E99 Action:T332
T333 Modifier 3961 3965 well
T334 Action 3966 3970 Load
E100 Action:T334 Acts-on:T336 Site:T338
T335 Modifier 3971 3979 purified
T336 Reagent 3980 3992 PCR products
T337 Concentration 4000 4002 6%
T338 Reagent 4003 4015 TBE-PAGE gel
T339 Amount 4264 4269 10 µL
T340 Location 4223 4237 adjacent lanes
T341 Reagent 4132 4139 Samples
T342 Action 4216 4219 run
E101 Action:T342 Site:T340 Using:E102
T343 Action 4190 4199 sequenced
E102 Action:T343 Acts-on:T346 Using:T347
T344 Action 4140 4148 prepared
E103 Action:T344 Acts-on:T341 Using:T345
T345 Method 4154 4172 different barcodes
T346 Mention 4177 4181 that
R1 Coreference-Link Arg1:T346 Arg2:T341
T347 Method 4200 4208 together
T348 Location 4245 4258 adjacent lane
T349 Action 4259 4263 load
E104 Action:T349 Acts-on:T350 Site:T348
T350 Reagent 4273 4300 Ready to Load Low MW Ladder
R2 Measure Arg1:T350 Arg2:T339
T351 Action 4302 4305 Run
E105 Action:T351 Acts-on:T352 Using:T354
T352 Reagent 4310 4313 gel
T353 Concentration 4319 4321 1X
T354 Reagent 4322 4332 TBE buffer
T355 Generic-Measure 4336 4341 200 V
T356 Reagent 4352 4366 lower dye band
T357 Location 4379 4385 bottom
T358 Reagent 4393 4396 gel
R3 Measure Arg1:T354 Arg2:T353
R4 Setting Arg1:E105 Arg2:T355
T359 Reagent 4413 4416 gel
T360 Modifier 4432 4445 approximately
T361 Time 4446 4456 30 minutes
T362 Action 4424 4427 run
E106 Action:T362 Acts-on:T359
R5 Setting Arg1:E106 Arg2:T361
R6 Mod-Link Arg1:T361 Arg2:T360
T363 Modifier 4512 4521 Carefully
T364 Action 4522 4528 remove
E107 Action:T364 Acts-on:T365 Site:T366
T365 Reagent 4533 4536 gel
T366 Location 4546 4558 glass plates
T367 Action 4563 4568 stain
E108 Action:T367 Using:T368 Using2:T370
T368 Reagent 4576 4594 nucleic acid stain
T369 Reagent 4603 4612 SYBR Gold
T370 Method 4630 4655 manufacturer instructions
T371 Reagent 4614 4624 Invitrogen
T372 Action 4657 4666 Visualize
E109 Action:T372 Acts-on:T373 Using:T374
T373 Reagent 4667 4676 gel bands
T374 Device 4682 4701 UV transilluminator
T375 Device 4711 4739 gel documentation instrument
T376 Modifier 4749 4754 clean
T377 Device 4755 4760 razor
T378 Reagent 4781 4785 band
T379 Action 4790 4795 place
E110 Action:T379 Acts-on:T382
T380 Modifier 4801 4806 clean
T381 Amount 4807 4813 1.7 mL
T382 Location 4814 4818 tube
T383 Action 4820 4834 Do not cut out
E111 Action:T383 Acts-on:T384
T384 Reagent 4839 4851 ~130 bp band
R7 Mod-Link Arg1:T377 Arg2:T376
T385 Action 4761 4768 cut out
E112 Action:T385 Using:T377 Acts-on:T378
T388 Generic-Measure 4773 4780 ~150 bp
R8 Measure Arg1:T378 Arg2:T388
T386 Modifier 5022 5029 Briefly
T387 Action 5030 5040 centrifuge
E113 Action:T387 Acts-on:T389
T389 Location 5045 5065 microcentrifuge tube
T390 Reagent 5081 5090 gel slice
T391 Action 5094 5101 collect
E114 Action:T391 Acts-on:T392 Site:T393 Using:E113
T392 Reagent 5106 5115 gel slice
T393 Location 5123 5129 bottom
T394 Location 5137 5141 tube
T395 Action 5143 5148 Crush
E115 Action:T395 Acts-on:T396 Using:T399
T396 Reagent 5153 5162 gel slice
T397 Modifier 5163 5173 thoroughly
T398 Modifier 5181 5191 disposable
T399 Device 5192 5198 pestle
T400 Action 5200 5205 Leave
E116 Action:T400 Acts-on:T401 Site:T402
T401 Device 5210 5216 pestle
T402 Location 5224 5228 tube
T403 Action 5230 5233 Add
E117 Action:T403 Acts-on:T405 Site:T407
T404 Amount 5234 5240 300 µL
T405 Reagent 5244 5258 Elution Buffer
T406 Modifier 5262 5266 each
T407 Location 5267 5271 tube
T408 Action 5281 5287 remove
E118 Action:T408 Acts-on:T409
T409 Device 5292 5298 pestle
T410 Reagent 5322 5325 gel
T411 Action 5347 5353 washed
E119 Action:T411 Site:T412 Acts-on:T410
T412 Device 5363 5369 pestle
T413 Time 5400 5407 2 hours
T414 Time 5411 5420 overnight
T415 Temperature 5424 5440 room temperature
T416 Method 5446 5455 agitation
T417 Modifier 5391 5399 at least
T418 Action 5386 5390 soak
E120 Action:T418 Acts-on:T419 Using:T416
T419 Reagent 5375 5385 gel pieces
T420 Action 5457 5472 DO NOT incubate
E121 Action:T420
T421 Time 5485 5494 overnight
T422 Action 5496 5501 Pulse
E122 Action:T422 Acts-on:T423
T423 Location 5502 5512 spin tubes
T424 Action 5516 5523 collect
E123 Action:T424 Acts-on:T426 Site:T427 Site2:T428 Using:E122
T425 Modifier 5524 5527 all
T426 Reagent 5528 5534 eluate
T427 Location 5540 5544 wall
T428 Seal 5549 5552 lid
R9 Mod-Link Arg1:T426 Arg2:T425
T429 Modifier 5554 5563 Carefully
T430 Action 5564 5572 transfer
E124 Action:T430 Acts-on:T431 Site:T432
T431 Reagent 5577 5583 eluate
T432 Location 5615 5618 top
T433 Location 5624 5645 Spin-X Centrifugetube
T434 Device 5680 5689 P1000 tip
T435 Action 5655 5662 Cutting
E125 Action:T435 Acts-on:T312
T437 Action 5702 5710 transfer
E126 Action:T437 Acts-on:T438
T438 Reagent 5714 5731 larger gel pieces
T439 Action 5733 5743 Centrifuge
E127 Action:T439 Acts-on:T440
T440 Location 5748 5759 Spin-X tube
T441 Speed 5763 5773 16,000 x g
T442 Time 5778 5787 2 minutes
T443 Action 5789 5796 Dispose
E128 Action:T443 Site:T444
T444 Device 5804 5815 spin filter
T445 Action 5817 5820 Add
E129 Action:T445 Acts-on:T447
T446 Modifier 5824 5828 each
T447 Location 5829 5833 tube
T448 Action 5838 5841 mix
E130 Action:T448 Acts-on:T450 Acts-on2:T452
T449 Amount 5849 5854 50 µL
T450 Reagent 5856 5871 AMPure XP Beads
T451 Amount 5873 5879 350 µL
T452 Reagent 5881 5892 Isopropanol
T453 Action 5895 5903 Incubate
E131 Action:T453 Acts-on:T455
T454 Temperature 5907 5923 room temperature
T455 Reagent 5928 5938 10 minutes
T456 Action 6010 6019 Magnetize
E132 Action:T456 Acts-on:T457
T457 Reagent 6020 6026 sample
T458 Reagent 6033 6041 solution
T459 Action 6050 6055 clear
E133 Action:T459 Acts-on:T458 Using:E132
T460 Modifier 6066 6075 Carefully
T461 Modifier 6178 6187 Carefully
T462 Action 6076 6082 remove
E134 Action:T462 Acts-on:T466
T463 Action 6188 6194 remove
E135 Action:T463 Acts-on:T467
T464 Action 6087 6094 discard
E136 Action:T464 Acts-on:T466
T465 Action 6199 6206 discard
E137 Action:T465 Acts-on:T467
T466 Reagent 6099 6110 supernatant
T467 Reagent 6211 6222 supernatant
T468 Action 6112 6115 add
E138 Action:T468 Acts-on:T474
T469 Action 6224 6227 add
E139 Action:T469 Acts-on:T475
T470 Amount 6116 6122 950 µL
T471 Amount 6228 6234 950 µL
T472 Concentration 6123 6126 80%
T473 Concentration 6235 6238 80%
T474 Reagent 6127 6134 ethanol
T475 Reagent 6239 6246 ethanol
T476 Action 6136 6144 incubate
E140 Action:T476
T477 Action 6248 6256 incubate
E141 Action:T477
T478 Time 6145 6155 30 seconds
T479 Time 6257 6267 30 seconds
T480 Action 6161 6167 remove
E142 Action:T480
T481 Action 6273 6279 remove
E143 Action:T481
R10 Mod-Link Arg1:E135 Arg2:T461
R11 Mod-Link Arg1:E134 Arg2:T460
R12 Measure Arg1:T474 Arg2:T472
R13 Measure Arg1:T475 Arg2:T473
R14 Measure Arg1:T475 Arg2:T471
R15 Measure Arg1:T474 Arg2:T470
T482 Action 6281 6284 Dry
E144 Action:T482 Acts-on:T483
T483 Reagent 6285 6291 sample
T484 Time 6296 6305 3 minutes
T485 Time 6313 6323 one minute
T486 Action 6325 6331 remove
E145 Action:T486 Acts-on:T487 Site:T488
T487 Reagent 6345 6351 liquid
T488 Location 6382 6388 bottom
T489 Location 6396 6400 tube
T490 Action 6402 6408 Remove
E146 Action:T490 Acts-on:T491 Site:T492
T491 Location 6409 6414 plate
T492 Device 6420 6433 magnetic rack
T493 Action 6438 6447 resuspend
E147 Action:T493 Acts-on:T494 Site:T496 Using:T497
T494 Reagent 6448 6459 bead pellet
T495 Amount 6463 6468 13 µL
T496 Reagent 6472 6491 Resuspension Buffer
T497 Method 6495 6523 pipetting volume up and down
T498 Reagent 6537 6542 beads
T499 Modifier 6547 6557 completely
T500 Action 6558 6569 resuspended
E148 Action:T500 Acts-on:T498
T501 Action 6586 6594 Incubate
E149 Action:T501
T502 Time 6599 6608 2 minutes
T503 Action 6610 6619 Magnetize
E150 Action:T503
T504 Time 6624 6633 5 minutes
T505 Reagent 6643 6654 supernatant
T506 Action 6663 6668 clear
E151 Action:T506 Acts-on:T505
T507 Action 6670 6678 Transfer
E152 Action:T507 Acts-on:T509 Site:T512
T508 Amount 6679 6684 12 µL
T509 Reagent 6688 6699 supernatant
T510 Modifier 6705 6710 clean
T511 Amount 6711 6717 1.7 mL
T512 Location 6718 6722 tube
R16 Mod-Link Arg1:T4 Arg2:T3
R17 Or Arg1:T7 Arg2:T8
R18 Meronym Arg1:T8 Arg2:T9
R19 Mod-Link Arg1:T13 Arg2:T12
R20 Mod-Link Arg1:T15 Arg2:T14
R21 Measure Arg1:T13 Arg2:T11
R22 Measure Arg1:T16 Arg2:T17
R23 Meronym Arg1:T16 Arg2:T15
R24 Measure Arg1:T19 Arg2:T18
R25 Meronym Arg1:T21 Arg2:T22
R26 Measure Arg1:T28 Arg2:T27
R27 Setting Arg1:E8 Arg2:T29
R28 Setting Arg1:E9 Arg2:T31
R29 Setting Arg1:E9 Arg2:T32
R30 Setting Arg1:E10 Arg2:T36
R31 Setting Arg1:E10 Arg2:T37
R32 Mod-Link Arg1:T45 Arg2:T44
R33 Mod-Link Arg1:T47 Arg2:T46
R34 Measure Arg1:T45 Arg2:T40
R35 Measure Arg1:T53 Arg2:T41
R36 Mod-Link Arg1:E13 Arg2:T51
T23 Action 637 640 Add
E6 Action:T23 Acts-on:T53
T513 Action 703 706 Add
E153 Action:T513 Acts-on:T54 Acts-on2:T55
R37 Measure Arg1:T54 Arg2:T42
R38 Measure Arg1:T55 Arg2:T43
R39 Mod-Link Arg1:E14 Arg2:T57
R40 Setting Arg1:E15 Arg2:T60
T514 Action 841 846 clear
E154 Action:T514 Acts-on:T63 Using:E16
R41 Measure Arg1:T75 Arg2:T73
R42 Measure Arg1:T76 Arg2:T74
R43 Mod-Link Arg1:T75 Arg2:T71
R44 Mod-Link Arg1:T76 Arg2:T72
R45 Measure Arg1:T75 Arg2:T68
R46 Measure Arg1:T76 Arg2:T69
R47 Setting Arg1:E21 Arg2:T79
R48 Setting Arg1:E22 Arg2:T80
R49 Or Arg1:T83 Arg2:T85
R50 Or Arg1:T84 Arg2:T86
R51 Setting Arg1:T83 Arg2:T87
R52 Setting Arg1:T84 Arg2:T88
R53 Setting Arg1:T86 Arg2:T88
R54 Setting Arg1:T85 Arg2:T87
R55 Setting Arg1:E25 Arg2:T91
R56 Mod-Link Arg1:T98 Arg2:T96
R57 Mod-Link Arg1:T98 Arg2:T97
R58 Meronym Arg1:T92 Arg2:T93
R59 Measure Arg1:T105 Arg2:T104
R60 Mod-Link Arg1:E30 Arg2:T108
R61 Setting Arg1:E31 Arg2:T111
R62 Measure Arg1:T114 Arg2:T113
R63 Mod-Link Arg1:T117 Arg2:T116
R64 Mod-Link Arg1:T117 Arg2:T115
R65 Measure Arg1:T124 Arg2:T123
R66 Measure Arg1:T127 Arg2:T126
R67 Mod-Link Arg1:E36 Arg2:T129
R68 Meronym Arg1:T127 Arg2:T125
R69 Measure Arg1:T133 Arg2:T132
R70 Measure Arg1:T136 Arg2:T135
R71 Mod-Link Arg1:E39 Arg2:T138
R72 Setting Arg1:E40 Arg2:T141
R73 Measure Arg1:T157 Arg2:T155
R74 Measure Arg1:T158 Arg2:T156
R75 Mod-Link Arg1:T157 Arg2:T153
R76 Mod-Link Arg1:T158 Arg2:T154
R77 Measure Arg1:T157 Arg2:T151
R78 Measure Arg1:T158 Arg2:T152
R79 Setting Arg1:E47 Arg2:T161
R80 Setting Arg1:E48 Arg2:T162
R81 Or Arg1:T165 Arg2:T167
R82 Or Arg1:T166 Arg2:T168
R83 Setting Arg1:T165 Arg2:T169
R84 Setting Arg1:T167 Arg2:T169
R85 Setting Arg1:T166 Arg2:T170
R86 Setting Arg1:T168 Arg2:T170
R87 Setting Arg1:E51 Arg2:T173
R88 Setting Arg1:E52 Arg2:T174
R89 Mod-Link Arg1:T178 Arg2:T177
R90 Mod-Link Arg1:T178 Arg2:T176
R91 Meronym Arg1:T180 Arg2:T181
R92 Measure Arg1:T188 Arg2:T187
R93 Mod-Link Arg1:E56 Arg2:T191
R94 Setting Arg1:E57 Arg2:T194
R95 Measure Arg1:T201 Arg2:T200
R96 Mod-Link Arg1:T203 Arg2:T202
R97 Measure Arg1:T206 Arg2:T205
R98 Setting Arg1:E62 Arg2:T208
R99 Setting Arg1:E62 Arg2:T209
R100 Mod-Link Arg1:E63 Arg2:T212
R101 Setting Arg1:E64 Arg2:T214
R102 Setting Arg1:E64 Arg2:T215
R103 Measure Arg1:T219 Arg2:T218
R104 Mod-Link Arg1:T221 Arg2:T220
R105 Setting Arg1:E66 Arg2:T223
R106 Setting Arg1:E66 Arg2:T224
R107 Mod-Link Arg1:E67 Arg2:T225
R108 Mod-Link Arg1:T228 Arg2:T229
R109 Mod-Link Arg1:E69 Arg2:T233
R110 Setting Arg1:E70 Arg2:T236
R111 Setting Arg1:E70 Arg2:T237
R112 Setting Arg1:E70 Arg2:T238
R113 Setting Arg1:E70 Arg2:T239
R114 Mod-Link Arg1:E72 Arg2:T245
R115 Setting Arg1:E71 Arg2:T242
R116 Measure Arg1:T251 Arg2:T249
R117 Mod-Link Arg1:T255 Arg2:T256
R118 Mod-Link Arg1:E75 Arg2:T259
R119 Measure Arg1:T252 Arg2:T250
R120 Measure Arg1:T254 Arg2:T253
R121 Mod-Link Arg1:E76 Arg2:T260
R122 Setting Arg1:E77 Arg2:T264
R123 Measure Arg1:T280 Arg2:T278
R124 Mod-Link Arg1:T280 Arg2:T276
R125 Measure Arg1:T281 Arg2:T279
R126 Measure Arg1:T280 Arg2:T274
R127 Mod-Link Arg1:T281 Arg2:T277
R128 Measure Arg1:T281 Arg2:T275
R129 Setting Arg1:E84 Arg2:T284
R130 Setting Arg1:E85 Arg2:T285
R131 Or Arg1:T288 Arg2:T290
R132 Or Arg1:T289 Arg2:T291
R133 Setting Arg1:T288 Arg2:T292
R134 Setting Arg1:T290 Arg2:T292
R135 Setting Arg1:T291 Arg2:T293
R136 Setting Arg1:T289 Arg2:T293
R137 Setting Arg1:E88 Arg2:T296
R138 Setting Arg1:E89 Arg2:T303
R139 Mod-Link Arg1:T300 Arg2:T299
R140 Mod-Link Arg1:T300 Arg2:T298
T515 Action 3611 3620 collected
E155 Action:T515 Creates:T300 Site:T301
R141 Meronym Arg1:T301 Arg2:T302
R142 Measure Arg1:T310 Arg2:T309
R143 Mod-Link Arg1:E93 Arg2:T314
R144 Setting Arg1:E94 Arg2:T317
R145 Measure Arg1:T323 Arg2:T322
R146 Mod-Link Arg1:T325 Arg2:T324
R147 Measure Arg1:T329 Arg2:T328
R148 Measure Arg1:T329 Arg2:T327
R149 Mod-Link Arg1:T331 Arg2:T330
R150 Mod-Link Arg1:E99 Arg2:T333
R151 Mod-Link Arg1:T336 Arg2:T335
R152 Measure Arg1:T338 Arg2:T337
R153 Mod-Link Arg1:E107 Arg2:T363
R154 Or Arg1:T368 Arg2:T369
R155 Or Arg1:T369 Arg2:T371
R156 Or Arg1:T374 Arg2:T375
R157 Measure Arg1:T382 Arg2:T381
R158 Mod-Link Arg1:T382 Arg2:T380
R159 Mod-Link Arg1:E113 Arg2:T386
R160 Meronym Arg1:T390 Arg2:T389
R161 Meronym Arg1:T393 Arg2:T394
R162 Mod-Link Arg1:E115 Arg2:T397
R163 Mod-Link Arg1:T399 Arg2:T398
R164 Measure Arg1:T405 Arg2:T404
R165 Mod-Link Arg1:T407 Arg2:T406
R166 Setting Arg1:E120 Arg2:T413
R167 Or Arg1:T413 Arg2:T414
R168 Mod-Link Arg1:T413 Arg2:T417
R169 Setting Arg1:E120 Arg2:T415
R170 Setting Arg1:E121 Arg2:T421
R171 Mod-Link Arg1:E124 Arg2:T429
R172 Meronym Arg1:T432 Arg2:T433
T312 Location 5667 5674 end off
R173 Meronym Arg1:T312 Arg2:T434
R174 Setting Arg1:E127 Arg2:T441
R175 Setting Arg1:E127 Arg2:T442
R176 Mod-Link Arg1:T447 Arg2:T446
T436 Modifier 5842 5846 well
R177 Mod-Link Arg1:E130 Arg2:T436
R178 Measure Arg1:T450 Arg2:T449
R179 Measure Arg1:T452 Arg2:T451
R180 Setting Arg1:E131 Arg2:T454
R181 Setting Arg1:E140 Arg2:T478
R182 Setting Arg1:E141 Arg2:T479
R183 Setting Arg1:E144 Arg2:T484
R184 Setting Arg1:E145 Arg2:T485
T516 Modifier 6336 6344 residual
T517 Modifier 6332 6335 all
R185 Mod-Link Arg1:T487 Arg2:T516
R186 Mod-Link Arg1:T487 Arg2:T517
R187 Meronym Arg1:T488 Arg2:T489
R188 Measure Arg1:T496 Arg2:T495
R189 Mod-Link Arg1:E148 Arg2:T499
R190 Setting Arg1:E149 Arg2:T502
R191 Setting Arg1:E150 Arg2:T504
R192 Measure Arg1:T509 Arg2:T508
R193 Measure Arg1:T512 Arg2:T511
R194 Mod-Link Arg1:T512 Arg2:T510