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XXXX-XX Bac Library: High Density Replica Filters (4x4 Array With Internal Duplicates)
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 been
spotted 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):
B F B C
A D G E
G H C F
Where A - H are 8 different clones
A E D H from 8 different plates
4. Each filter is spotted in 6 different panels which contain 8 plates each, spotted twice:
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.
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
10 34 10 16 8 32 8 14
4 22 40 28 2 20 38 26
40 46 16 34 38 44 14 32
4 28 22 46 2 26 20 44
Panel 1 Panel 6
7 31 7 13 12 36 12 18
1 19 37 25 6 24 42 30
37 43 13 31 42 48 18 36
1 25 19 43 6 30 24 48
Panel 5 Panel 3
11 35 11 17 9 33 9 15
5 23 41 29 3 21 39 27
41 47 17 35 39 45 15 33
5 29 23 47 3 27 21 45
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
193-240
241-288
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 is
derived. See next page for vector patterns seen on filters.
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 with
the date and filter number at the top, each panel is divided up into rows and columns as follows:
P
O
N
M
L
K
J
I
H
G
F
E
D
C
B
A
24 23 22 21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1
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, in
this example the positive is located in plate #222 at C-22.
Examples of Positive Clone Location in Original 384-well plates
Vector Sheet
Vectors
PlatesPlatesPlatesPlatesPlatesPlatesPlatesPlatesSegment 111-67-1213-1819-2425-3031-3637-4243-
Filter #
48249-5455-6061-6667-7273-7879-8485-9091-96397-102103-108109-114115-120121-126127-132133-138139-
1444145-150151-156157-162163-168169-174175-180181-186187-1925193-198199-204205-210211-216217-
222223-228229-234235-2406241-246247-252253-258259-264265-270271-276277-282283-288Segment 27289-
294295-300301-306307-312313-318319-324325-330331-3368337-342343-348349-354355-360361-366367-372373-
378379-3849385-390391-396397-402403-408409-414415-420421-426427-43210433-438439-444445-450451-
456457-462463-468469-474475-48011481-486487-492493-498499-504505-510511-516517-522523-52812529-
534535-540541-546547-552553-558559-564565-570571-576
Vector Sheet
Vectors
Vectors
31
32
33
34
35
36
Plate locator Sheet – XXXX-XX
Segment #1
Filter 1 Filter 2
2 2 8 14 20 26 32 38 44 2 50 56 62 68 74 80 86 92
3 3 9 15 21 27 33 39 45 3 51 57 63 69 75 81 87 93
4 4 10 16 22 28 34 40 46 4 52 58 64 70 76 82 88 94
5 5 11 17 23 29 35 41 47 5 53 59 65 71 77 83 89 95
6 6 12 18 24 30 36 42 48 6 54 60 66 72 78 84 90 96
Filter 3 Filter 4
2 98 104 110 116 122 128 134 140 2 146 152 158 164 170 176 182 188
3 99 105 111 117 123 129 135 141 3 147 153 159 165 171 177 183 189
4 100 106 112 118 124 130 136 142 4 148 154 160 166 172 178 184 190
5 101 107 113 119 125 131 137 143 5 149 155 161 167 173 179 185 191
6 102 108 114 120 126 132 138 144 6 150 156 162 168 174 180 186 192
Filter 5 Filter 6
Filter 7 Filter 8
2 290 296 302 308 314 320 326 332 2 338 344 350 356 362 368 374 380
3 291 297 303 309 315 321 327 333 3 339 345 351 357 363 369 375 381
4 292 298 304 310 316 322 328 334 4 340 346 352 358 364 370 376 382
5 293 299 305 311 317 323 329 335 5 341 347 353 359 365 371 377 383
6 294 300 306 312 318 324 330 336 6 342 348 354 360 366 372 378 384
Filter 9 Filter 10
2 386 392 398 404 410 416 422 428 2 434 440 446 452 458 464 470 476
3 387 393 399 405 411 417 423 429 3 435 441 447 453 459 465 471 477
4 388 394 400 406 412 418 424 430 4 436 442 448 454 460 466 472 478
5 389 395 401 407 413 419 425 431 5 437 443 449 455 461 467 473 479
6 390 396 402 408 414 420 426 432 6 438 444 450 456 462 468 474 480
Filter 11 Filter 12
Filter 13 Filter 14
2 578 584 590 596 602 608 614 620 2 626 632 638 644 650 656 662 668
3 579 585 591 597 603 609 615 621 3 627 632 639 645 651 657 663 669
4 580 586 592 598 604 610 616 622 4 628 632 640 646 652 658 664 670
5 581 587 593 599 605 611 617 623 5 629 632 641 647 653 659 665 671
6 582 588 594 600 606 612 618 624 6 630 632 642 648 654 660 666 672
Filter 15 Filter 16
2 674 680 686 692 698 704 710 716 2 722 728 734 740 746 752 758 764
3 675 681 687 693 699 705 711 717 3 723 729 735 741 747 753 759 765
4 676 682 688 694 700 706 712 718 4 724 730 736 742 748 754 760 766
5 677 683 689 695 701 707 713 719 5 725 731 737 743 749 755 761 767
6 678 684 690 696 702 708 714 720 6 726 732 738 744 750 756 762 768
Filter 17 Filter 18
Filter 19 Filter 20
2 866 872 878 884 890 896 902 908 2 914 920 926 932 938 944 950 956
3 867 873 879 885 891 897 903 909 3 915 921 927 933 939 945 951 957
4 868 874 880 886 892 898 904 910 4 916 922 928 934 940 946 952 958
5 869 875 881 887 893 899 905 911 5 917 923 929 935 941 947 953 959
6 870 876 882 888 894 900 906 912 6 918 924 930 936 942 948 954 960
Filter 21 Filter 22
2 962 968 974 980 986 992 998 1004 2 1010 1016 1022 1028 1034 1040 1046 1052
3 963 969 975 981 987 993 999 1005 3 1011 1017 1023 1029 1035 1041 1047 1053
4 964 970 976 982 988 994 1000 1006 4 1012 1018 1024 1030 1036 1042 1048 1054
5 965 971 977 983 989 995 1001 1007 5 1013 1019 1025 1031 1037 1043 1049 1055
6 966 972 978 984 990 996 1002 1008 6 1014 1020 1026 1032 1038 1044 1050 1056
Filter 23 Filter 24
Filter 25 Filter 26
2 1154 1160 1166 1172 1178 1184 1190 1196 2 1202 1208 1214 1220 1226 1232 1238 1244
3 1155 1161 1167 1173 1179 1185 1191 1197 3 1203 1209 1215 1221 1227 1233 1239 1245
4 1156 1162 1168 1174 1180 1186 1192 1198 4 1204 1210 1216 1222 1228 1234 1240 1246
5 1157 1163 1169 1175 1181 1187 1193 1199 5 1205 1211 1217 1223 1229 1235 1241 1247
6 1158 1164 1170 1176 1182 1188 1194 1200 6 1206 1212 1218 1224 1230 1236 1242 1248
Filter 27 Filter 28
2 1250 1256 1262 1268 1274 1280 1286 1292 2 1298 1304 1310 1316 1322 1328 1334 1340
3 1251 1257 1263 1269 1275 1281 1287 1293 3 1299 1305 1311 1317 1323 1329 1335 1341
4 1252 1258 1264 1270 1276 1282 1288 1294 4 1300 1306 1312 1318 1324 1330 1336 1342
5 1253 1259 1265 1271 1277 1283 1289 1295 5 1301 1307 1313 1319 1325 1331 1337 1343
6 1254 1260 1266 1272 1278 1284 1290 1296 6 1302 1308 1314 1320 1326 1332 1338 1344
Filter 29 Filter 30