Hybridization Instructions

8/8/2019 Hybridization Instructions

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XXXX-XX BAC LIBRARY

High Density Replica Filters

(4x4 array with internal duplicates)

1. Each filter contains 36,864 colonies which represent 18,432 independent clones that have beenspotted in duplicate.

2. On each filter there are 6 fields consisting of 16x24 boxes containing 16 colonies each (8 clones

spotted in duplicate). Each filter therefore has 48x48 boxes of 16 clones.

3. Within each box of 16 colonies, there are 8 different duplicate pair orientations (called vectors):

Where A - H are 8 different clonesfrom 8 different plates

4. Each filter is spotted in 6 different panels which contain 8 plates each, spotted twice:

Date Code Filter Number

Panel 4 Panel 2

Panel 1 Panel 6

Panel 5 Panel 3

Each panel consists of 24 x 16 boxes of 16 colonies each. There are 384 boxes in each panel,

each corresponding to the plate wells.

B F B C

A D G E

G H C F

A E D H


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Determining Location of Positive Clone in Original 384-well Plates

1. Hold filter autorad with date at the top left hand side, and the filter number at the top right hand

side. Place overlay template on op of the autorad using the background colonies as an alignment

guide.

2. Determine which panel the positive clone is located in.3. Plate #l is spotted in panel 1, plate #2 is spotted in panel 2, etc., then plate #7 is spotted in panel

1, plate #8 is in panel 2, etc.

4. The first 48 plates of the library are spotted as follows:

Panel 4 Panel 2

Panel 1 Panel 6

Panel 5 Panel 3

Each group of numbers above represents one box of 16 colonies out of 384 boxes in each panel.

They therefore show the 384-well plates that have been spotted in each panel.Filters containing plates: 49-96

97-144

145-192

193-240241-288

289-336 are spotted in an analogous manner.

5. The orientation of the positive signal (vector) gives the 384-well plate from which the clone isderived. See next page for vector patterns seen on filters.

8 32 8 14

2 20 38 26

38 44 14 32

2 26 20 44

10 34 10 16

4 22 40 28

40 46 16 34

4 28 22 46

12 36 12 18

6 24 42 30

42 48 18 36

6 30 24 48

7 31 7 13

1 19 37 25

37 43 13 31

1 25 19 43

11 35 11 17

5 23 41 29

41 47 17 35

5 29 23 47

9 33 9 15

3 21 39 27

39 45 15 33

3 27 21 45


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Determining the Row and Column of the 384-Well Plate a Clone is Derived From

The 24 x 16 boxes in each panel correspond to the format of the 384-well plate. Holding the filter withthe date and filter number at the top, each panel is divided up into rows and columns as follows:

PONMLKJIHGFEDCB

A24 23 22 21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1

In summary, to locate a positive clone in the original 384-well plate:

First note which filter the positive is on and which plates are spotted on the filter, then:

a) Place overlay template over autorad of filter using the background spots as an alignmentguide.

b) Determine which panel the positive is in.

c) Determine plate number range from the vector direction using the vector sheet provided.d) Determine the exact plate number using the Plate Locator Sheet provided. Find the plate

number that falls in the range of plates determined above for the specific filter and panel

number of interest.e) Using the overlay template determine the well coordinates of the positive.


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Examples of Positive Clone Location Determination

The following examples refer to the photocopy of an autorad of a Human BAC 4x4 high density filter(32P-labeled probe) which has been overlaid with the transparent template. The positive duplicate spots

are circled and marked A, B, or C.

Example I: Positive A.

In this example assume the filter is number 1.

a) The positive duplicate spots are in panel 6.b) Determine the vector (orientation of the duplicate positive spots) direction and find that

pattern on the Vector Sheet. Since the positive is on fitter 1, we now know that it is located in

plates #1-6.c) Using the Plate Locator Sheet, we determine that plate #6 is the only plate in the range of #1

thru #6 that is spotted in panel 6 on fitter 1.

d) Reading directly off the autorad overlaid with the template, we determine that the plate

coordinates are E-5. Therefore, the positive clone in this example is in plate #6 at E-5.

Example 2: Positive B.

In this example assume that the filter is number 3.a) The positive duplicate spots are in panel 2.

b) Using the Vector Sheet, the vector direction tells us that the positive is in plates #127-l32

(since we know that the fitter is #3)c) Using the Plate Locator Sheet, we determine that plate #128 is the Plate number within the

range of #127-132 that is located on fitter 3 in panel 2.

d) Reading directly from the template, the positive is in well J-14. Therefore, the positive clone

in this example in is plate #128 at J-14.

Example 3: Positive C.In this example assume that the filter is number 5.

a) The positive duplicate spots are in panel 6

b) On the Vector Sheet, the orientation of the duplicate positive spots tell us that it is located in

plates #217-222 on fitter #5.c) On the Plate Locator Sheet, we determine that plate #222 is the one in the range of #217-222

that is gridded in panel 6 of filter #5.

d) Reading directly from the overlay template, the positive is at coordinate C-22, Therefore, inthis example the positive is located in plate #222 at C-22.


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Examples of Positive Clone Location in Original 384-well plates


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Vector Sheet

Date Barcode Number XXXX-XX Filter Informati

Vectors

Filter #PlatesPlatesPlatesPlatesPlatesPlatesPlatesPlatesSegment 111-67-1213-1819-2425-3031-3637-4243-

48249-5455-6061-6667-7273-7879-8485-9091-96 397-102103-108109-114115-120121-126127-132133-138139-

1444145-150151-156157-162163-168169-174175-180181-186187-192 5193-198199-204205-210211-216217-222223-228229-234235-2406241-246247-252253-258259-264265-270271-276277-282283-288 Segment 27289

294295-300301-306307-312313-318319-324325-330331-336 8337-342343-348349-354355-360361-366367-37237

378379-3849385-390391-396397-402403-408409-414415-420421-426427-432 10433-438439-444445-450451-

456457-462463-468469-474475-480 11481-486487-492493-498499-504505-510511-516517-522523-528 12529534535-540541-546547-552553-558559-564565-570571-576


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Vector Sheet

Date Barcode Number XXXX-XX Filter Informatio

Vectors

Filter # Plates Plates Plates Plates Plates Plates Plates Plates

Segment3

13 577-582 583-588 589-594 595-600 601-606 607-612 613-618 619-624

14 625-630 631-636 637-642 643-648 649-654 655-660 661-666 667-672

15 673-678 679-684 685-690 691-696 697-702 703-708 709-714 715-720

16 721-726 727-732 733-738 739-744 745-750 751-756 757-762 763-768

17 769-774 775-780 781-786 787-792 793-798 799-804 805-810 811-816

18 817-822 823-828 829-834 835-840 841-846 847-852 853-858 859-864

Segment4

19 865-870 871-876 877-882 883-888 889-894 895-900 901-906 907-912

20 913-918 919-924 925-930 931-936 937-942 943-948 949-954 955-960

21 961-966 967-972 973-978 979-984 985-990 991-996 997-1002 1003-100

22 1009-1014 1015-1020 1021-1026 1027-1032 1033-1038 1039-1044 1045-1050 1051-105

23 1057-1062 1063-1068 1069-1074 1075-1080 1081-1086 1087-1092 1093-1098 1099-110

24 1105-1110 1111-1116 1117-1122 1123-1128 1129-1134 1135-1140 1140-1146 1147-115


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Vector Sheet

Date Barcode Number XXXX-XX Filter Informatio

Vectors

Filter # Plates Plates Plates Plates Plates Plates Plates Plates

Segment5

25 1153-1158 1159-1164 1165-1170 1171-1176 1177-1182 1183-1188 1189-1194 1195-120

26 1201-1206 1207-1212 1213-1218 1219-1224 1225-1230 1231-1236 1237-1242 1243-124

27 1249-1254 1255-1260 1261-1266 1267-1272 1273-1278 1279-1284 1285-1290 1291-129

28 1297-1302 1303-1308 1309-1314 1315-1320 1321-1326 1327-1332 1333-1338 1339-134

29 1345-1350 1351-1356 1357-1362 1363-1368 1369-1374 1375-1380 1381-1386 1387-139

30 1393-1398 1399-1404 1405-1410 1411-1416 1417-1422 1423-1428 1429-1434 1435-144

Segment6

31

32

33

34

35

36


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Plate locator Sheet XXXX-XX

Segment #1

Filter 1 Filter 2

Filter 3 Filter 4

Filter 5 Filter 6

Panel Plates

1 1 7 13 19 25 31 37 43

2 2 8 14 20 26 32 38 44

3 3 9 15 21 27 33 39 45

4 4 10 16 22 28 34 40 46

5 5 11 17 23 29 35 41 47

6 6 12 18 24 30 36 42 48

Panel Plates

1 49 55 61 67 73 79 85 91

2 50 56 62 68 74 80 86 92

3 51 57 63 69 75 81 87 93

4 52 58 64 70 76 82 88 94

5 53 59 65 71 77 83 89 95

6 54 60 66 72 78 84 90 96

Panel Plates

1 145 151 157 163 169 175 181 187

2 146 152 158 164 170 176 182 188

3 147 153 159 165 171 177 183 189

4 148 154 160 166 172 178 184 190

5 149 155 161 167 173 179 185 191

6 150 156 162 168 174 180 186 192

Panel Plates

1 97 103 109 115 121 127 133 139

2 98 104 110 116 122 128 134 140

3 99 105 111 117 123 129 135 141

4 100 106 112 118 124 130 136 142

5 101 107 113 119 125 131 137 143

6 102 108 114 120 126 132 138 144

Panel Plates

1 241 247 253 259 265 271 277 283

2 242 248 254 260 266 272 278 284

3 243 249 255 261 267 273 279 285

4 244 250 256 262 268 274 280 286

5 245 251 257 263 269 275 281 287

6 246 252 258 264 270 276 282 288

Panel Plates

1 193 199 205 211 217 223 229 235

2 194 200 206 212 218 224 230 236

3 195 201 207 213 219 225 231 237

4 196 202 208 214 220 226 232 238

5 197 203 209 215 221 227 233 239

6 198 204 210 216 222 228 234 240


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Segment #2

Filter 7 Filter 8

Filter 9 Filter 10

Filter 11 Filter 12

Panel Plates

1 289 295 301 307 313 319 325 331

2 290 296 302 308 314 320 326 332

3 291 297 303 309 315 321 327 333

4 292 298 304 310 316 322 328 334

5 293 299 305 311 317 323 329 335

6 294 300 306 312 318 324 330 336

Panel Plates

1 337 343 349 355 361 367 373 379

2 338 344 350 356 362 368 374 380

3 339 345 351 357 363 369 375 381

4 340 346 352 358 364 370 376 382

5 341 347 353 359 365 371 377 383

6 342 348 354 360 366 372 378 384

Panel Plates

1 433 439 445 451 457 463 469 475

2 434 440 446 452 458 464 470 476

3 435 441 447 453 459 465 471 477

4 436 442 448 454 460 466 472 478

5 437 443 449 455 461 467 473 479

6 438 444 450 456 462 468 474 480

Panel Plates

1 385 391 397 403 409 415 421 427

2 386 392 398 404 410 416 422 428

3 387 393 399 405 411 417 423 429

4 388 394 400 406 412 418 424 430

5 389 395 401 407 413 419 425 431

6 390 396 402 408 414 420 426 432

Panel Plates

1 529 535 541 547 553 559 565 571

2 530 536 542 548 554 560 566 572

3 531 537 543 549 555 561 567 573

4 532 538 544 550 556 562 568 574

5 533 539 545 551 557 563 569 575

6 534 540 546 552 558 564 570 576

Panel Plates

1 481 487 493 499 505 511 517 523

2 482 488 494 500 506 512 518 524

3 483 489 495 501 507 513 519 525

4 484 490 496 502 508 514 520 526

5 485 491 497 503 509 515 521 527

6 486 492 498 504 510 516 522 528


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Segment #3

Filter 13 Filter 14

Filter 15 Filter 16

Filter 17 Filter 18

Panel Plates

1 577 583 589 595 601 607 613 619

2 578 584 590 596 602 608 614 620

3 579 585 591 597 603 609 615 621

4 580 586 592 598 604 610 616 622

5 581 587 593 599 605 611 617 623

6 582 588 594 600 606 612 618 624

Panel Plates

1 625 632 637 643 649 655 661 667

2 626 632 638 644 650 656 662 668

3 627 632 639 645 651 657 663 669

4 628 632 640 646 652 658 664 670

5 629 632 641 647 653 659 665 671

6 630 632 642 648 654 660 666 672

Panel Plates

1 721 727 733 739 745 751 757 763

2 722 728 734 740 746 752 758 764

3 723 729 735 741 747 753 759 765

4 724 730 736 742 748 754 760 766

5 725 731 737 743 749 755 761 767

6 726 732 738 744 750 756 762 768

Panel Plates

1 673 679 685 691 697 703 709 715

2 674 680 686 692 698 704 710 716

3 675 681 687 693 699 705 711 717

4 676 682 688 694 700 706 712 718

5 677 683 689 695 701 707 713 719

6 678 684 690 696 702 708 714 720

Panel Plates

1 817 823 829 835 841 847 853 859

2 818 824 830 836 842 848 854 860

3 819 825 831 837 843 849 855 861

4 820 826 832 838 844 850 856 862

5 821 827 833 839 845 851 857 863

6 822 828 834 840 846 852 858 864

Panel Plates

1 769 775 781 787 793 799 805 811

2 770 776 782 788 794 800 806 812

3 771 777 783 789 795 801 807 813

4 772 778 784 790 796 802 808 814

5 773 779 785 791 797 803 809 815

6 774 780 786 792 798 804 810 816


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Segment #4

Filter 19 Filter 20

Filter 21 Filter 22

Filter 23 Filter 24

Panel Plates

1 865 871 877 883 889 895 901 907

2 866 872 878 884 890 896 902 908

3 867 873 879 885 891 897 903 909

4 868 874 880 886 892 898 904 910

5 869 875 881 887 893 899 905 911

6 870 876 882 888 894 900 906 912

Panel Plates

1 913 919 925 931 937 943 949 955

2 914 920 926 932 938 944 950 956

3 915 921 927 933 939 945 951 957

4 916 922 928 934 940 946 952 958

5 917 923 929 935 941 947 953 959

6 918 924 930 936 942 948 954 960

Panel Plates

1 1009 1015 1021 1027 1033 1039 1045 1051

2 1010 1016 1022 1028 1034 1040 1046 1052

3 1011 1017 1023 1029 1035 1041 1047 1053

4 1012 1018 1024 1030 1036 1042 1048 1054

5 1013 1019 1025 1031 1037 1043 1049 1055

6 1014 1020 1026 1032 1038 1044 1050 1056

Panel Plates

1 961 967 973 979 985 991 997 1003

2 962 968 974 980 986 992 998 1004

3 963 969 975 981 987 993 999 1005

4 9 64 970 976 98 2 988 99 4 1000 100 6

5 9 65 971 977 98 3 989 99 5 1001 100 7

6 9 66 972 978 98 4 990 99 6 1002 100 8

Panel Plates

1 1105 1111 1117 1123 1129 1135 1141 1147

2 1106 1112 1118 1124 1130 1136 1142 1148

3 1107 1113 1119 1125 1131 1137 1143 1149

4 1108 1114 1120 1126 1132 1138 1144 1150

5 1109 1115 1121 1127 1133 1139 1145 1151

6 1110 1116 1122 1128 1134 1140 1146 1152

Panel Plates

1 1057 1063 1069 1075 1081 1087 1093 1099

2 1058 1064 1070 1076 1082 1088 1094 1100

3 1059 1065 1071 1077 1083 1089 1095 1101

4 1060 1066 1072 1078 1084 1090 1096 1102

5 1061 1067 1073 1079 1085 1091 1097 1103

6 1062 1068 1074 1080 1086 1092 1098 1104


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Segment #5

Filter 25 Filter 26

Filter 27 Filter 28

Filter 29 Filter 30

Panel Plates

1 1153 1159 1165 1171 1177 1183 1189 1195

2 1154 1160 1166 1172 1178 1184 1190 1196

3 1155 1161 1167 1173 1179 1185 1191 1197

4 1156 1162 1168 1174 1180 1186 1192 1198

5 1157 1163 1169 1175 1181 1187 1193 1199

6 1158 1164 1170 1176 1182 1188 1194 1200

Panel Plates

1 1201 1207 1213 1219 1225 1231 1237 1243

2 1202 1208 1214 1220 1226 1232 1238 1244

3 1203 1209 1215 1221 1227 1233 1239 1245

4 1204 1210 1216 1222 1228 1234 1240 1246

5 1205 1211 1217 1223 1229 1235 1241 1247

6 1206 1212 1218 1224 1230 1236 1242 1248

Panel Plates

1 1297 1303 1309 1315 1321 1327 1333 1339

2 1298 1304 1310 1316 1322 1328 1334 1340

3 1299 1305 1311 1317 1323 1329 1335 1341

4 1300 1306 1312 1318 1324 1330 1336 1342

5 1301 1307 1313 1319 1325 1331 1337 1343

6 1302 1308 1314 1320 1326 1332 1338 1344

Panel Plates

1 1249 1255 1261 1267 1273 1279 1285 1291

2 1250 1256 1262 1268 1274 1280 1286 1292

3 1251 1257 1263 1269 1275 1281 1287 1293

4 1252 1258 1264 1270 1276 1282 1288 1294

5 1253 1259 1265 1271 1277 1283 1289 1295

6 1254 1260 1266 1272 1278 1284 1290 1296

Panel Plates

1 1393 1399 1405 1411 1417 1423 1429 1435

2 1394 1400 1406 1412 1418 1424 1430 1436

3 1395 1401 1407 1413 1419 1425 1431 1437

4 1396 1402 1408 1414 1420 1426 1432 1438

5 1397 1403 1409 1415 1421 1427 1433 1439

6 1398 1404 1410 1416 1422 1428 1434 1440

Panel Plates

1 1345 1351 1357 1363 1369 1375 1381 1387

2 1346 1352 1358 1364 1370 1376 1382 1388

3 1347 1353 1359 1365 1371 1377 1383 1389

4 1348 1354 1360 1366 1372 1378 1384 1390

5 1349 1355 1361 1367 1373 1379 1385 1391

6 1350 1356 1362 1368 1374 1380 1386 1392

Hybridization Instructions

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