Repeat Digest Protocol
Some digest will need to be repeated, as quantification of DNA was missed prior to digestion. Plans for each plate are below:
86 samples need to be cleaned.
Count | Client Plate Name | GTL Plate Name | Low Volume Wells | Empty Wells | Repeat RD Protocol | Dirty Sample Wells | New Normalization Made |
|---|---|---|---|---|---|---|---|
1 | MT_WCT_plate15 | WR_WCT15 |
| E8-H8, Col 9, 10, 11, 12 | *only 9 samples on plate and all need dilution. Add 15uL of AE to E7, B8, Add 30uL of AE to D7, G7, H7, A8, C8, D8 Add 45uL of AE to F7, | None | Dilutions done on org |
2 | MT_YCT_RangewideRADs_plate01 | WR_MYR1 |
|
| 81 samples need dilution - normalize 140ng/uL | None | Yes |
3 | MT_YCT_RangewideRADs_plate03 | WR_MYR3 |
|
| 89 samples need dilution - normalize 140ng/uL | None | Yes |
4 | Wrosenthal_GY21_YCT_tray1 | WR_GYCT1 | G1, H1, C9, F9, H9, F10, A12-D12 |
| 61 samples need dilution - normalize to 140ng/uL | A1-E1, C4, E4, E5, C7, E7 (10 samples) | Yes |
5 | Wrosenthal_GY21_YCT_tray3 | WR_GYCT3 | H1 |
| 40 samples need dilution - normalize to 140ng/uL | None | Yes |
6 | Wrosenthal_GY21_YCT_tray4 | WR_GYCT4 | A10 |
| 43 samples need dilution - normalize to 140ng/uL | F12, G12 | Yes |
7 | Wrosenthal_GY21_YCT_tray6 | WR_GYCT6 |
|
| 74 samples need dilution - normalize to 140ng/uL | None | Yes |
8 | Wrosenthal_GY21_YCT_tray7 | WR_GYCT7 |
| H8, Col 9, Col 10, Col 11, A12-G12 | 48 samples need dilution - normalize to 140ng/uL | None | Yes |
9 | Wrosenthal_PM_YCT_plate1 | WR_PMCT1 |
| H4, Col 5, A6-E6 | Add 15uL of AE to C1, F1, H1, A2 Add 30uL of AE to E1 Add to new plate | None | No |
10 | Wrosenthal_PM_YCT_plate2 | WR_PMCT2 | H4, B8 |
| 54 samples need dilution - normalize to 140ng/uL | H4 | Yes |
11 | WRosenthal_Trout_plate10 | WRT10 |
|
| Add 15uL of AE to D2, F2, C3, F4, B5, D6, F6 and add to new plate | None | No |
12 | WRosenthal_Trout_plate11 | WRT11 |
|
| N/A | None |
|
13 | WRosenthal_Trout_plate12 | WRT12 |
|
| Add well F5 to new plate (was empty, double check wksht) Add 15uL to E2, E10, G11 and add to new plate | E3, A4 |
|
14 | WRosenthal_Trout_plate13 | WRT13 |
| B8 | Add 15uL to F3, H6, F7, H8, F9, H9, and add to new plate. | E7 |
|
15 | WRosenthal_Trout_plate14 | WRT14 |
|
| Add 15uL to E2, C5, D7, F8, G8, C9, D10 | None |
|
16 | WRosenthal_Trout_plate15 | WRT15 |
|
| Add 15uL to E1-G1, D2, E2, C3, F3, G4, D5, E5, C8, C9-E9, C11 | D5 |
|
17 | WRosenthal_Trout_plate16 | WRT16 |
|
| Add 15uL to F2, A7, D7, B10 | None |
|
18 | WRosenthal_Trout_plate17 | WRT17 |
| D5, F5 | Add H6 to new plate (was empty) Add 15uL to B1, C1, E1, F1, G2, E6, G6, C7, F7, H7, B9, A10, D10, B11, D11, G12 and add to new plate
| D3 |
|
19 | WRosenthal_Trout_plate18 | WRT18 |
|
| Add 15uL to C1, D1, E1, F9 | None |
|
20 | WRosenthal_Trout_plate19 | WRT19 |
|
| Add 15uL to A2, C2, F2, H3, E4, G4, H6, D7, F7-H7, B8, D8, F9, A10, C10, D10, F10, H10, G11, C12, D12, G12 | D8 |
|
21 | WRosenthal_Trout_plate20 | WRT20 |
|
| 34 samples need dilution - normalize to 140ng/uL | None | Yes |
22 | WRosenthal_Trout_plate21 | WRT21 |
| G11, H11 | 44 samples need dilution - normalize to 140ng/uL | F1, G4 | Yes |
23 | WRosenthal_Trout_plate22 | WRT22 |
|
| 34 samples need dilution - normalize to 140ng/uL | D5, E5, A6-C6, A7 (6 samples) | Yes |
24 | WRosenthal_Trout_plate24 | WRT24 |
| H6, G12, H12 | 82 samples need dilution - normalize to 140ng/uL | F1, G1, F3, B4-G4, B5, D5, E5, C6, F6, A9, B9, F9, B10-F10, A12, D12 (24 samples) | Yes |
25 | WRosenthal_Trout_plate25* *Use A7 and B7 from original digest for ligation
| WRT25 |
| C6-H6, H9, Col 10, 11, 12 | 42 samples need dilution - normalize to 140ng/uL | A1, C1, E1, H1, G2, H2, F3-H3, A4, D4, E4, G4, H4, B5, H7, B9, E9 (18 samples) | Yes |
26 | WRosenthal_Trout_plate9 | WRT9 |
|
| Add 15uL of AE to A1, B1, F6, and B8 | A7 |
|
27 | WY_YCT_2021_plate04 | WR_WYCT4 |
| E12, F12 | 87 samples need dilution - normalize to 140ng/uL | B9 | Yes |
28 | WY_YCT_2021_plate09 | WR_WYCT9 |
|
| 77 samples need dilution - normalize to 140ng/uL | F8 | Yes |
29 | WY_YCT_2021_plate10 | WR_WYCT10 |
|
| 91 samples need dilution - normalize to 140ng/uL | C9-F9, H9, A10, C10, F10, G10, C11, D11, F11-H11 (14 samples) | Yes |
30 | WY_YCT_2021_plate11 | WR_WYCT11 |
|
| 77 samples need dilution - normalize to 140ng/uL | None | Yes |
31 | WY_YCT_2021_plate13 | WR_WYCT13 |
|
| 40 samples need dilution - normalize to 140ng/uL | None | Yes |
32 | WY_YCT_2021_plate14 | WR_WYCT14 |
| Col 11, 12 | 50 samples need dilution - normalize to 140ng/uL | None | Yes |
Count | Client Plate Name | GTL Plate Name | Low Volume Wells | Empty Wells | Repeat RD Protocol | Dirty Sample Wells | New Normalization Made |
|---|---|---|---|---|---|---|---|
1 | MT_WCT_plate15 | WR_WCT15 |
| E8-H8, Col 9, 10, 11, 12 | *only 9 samples on plate and all need dilution. Add 15uL of AE to E7, B8, Add 30uL of AE to D7, G7, H7, A8, C8, D8 Add 45uL of AE to F7, | None | Dilutions done on org |
4 | Wrosenthal_GY21_YCT_tray1 | WR_GYCT1 | G1, H1, C9, F9, H9, F10, A12-D12 |
| 61 samples need dilution - normalize to 140ng/uL | A1-E1, C4, E4, E5, C7, E7 (10 samples) | Yes |
5 | Wrosenthal_GY21_YCT_tray3 | WR_GYCT3 | H1 |
| 40 samples need dilution - normalize to 140ng/uL | None | Yes |
6 | Wrosenthal_GY21_YCT_tray4 | WR_GYCT4 | A10 |
| 43 samples need dilution - normalize to 140ng/uL | F12, G12 | Yes |
7 | Wrosenthal_GY21_YCT_tray6 | WR_GYCT6 |
|
| 74 samples need dilution - normalize to 140ng/uL | None | Yes |
8 | Wrosenthal_GY21_YCT_tray7 | WR_GYCT7 |
| H8, Col 9, Col 10, Col 11, A12-G12 | 48 samples need dilution - normalize to 140ng/uL | None | Yes |
9 | Wrosenthal_PM_YCT_plate1 | WR_PMCT1 |
| H4, Col 5, A6-E6 | Add 15uL of AE to C1, F1, H1, A2 Add 30uL of AE to E1 Add to new plate | None | No |
21 | WRosenthal_Trout_plate20 | WRT20 |
|
| 34 samples need dilution - normalize to 140ng/uL | None | Yes |
23 | WRosenthal_Trout_plate22 | WRT22 |
|
| 34 samples need dilution - normalize to 140ng/uL | D5, E5, A6-C6, A7 (6 samples) | Yes |
24 | WRosenthal_Trout_plate24 | WRT24 |
| H6, G12, H12 | 82 samples need dilution - normalize to 140ng/uL | F1, G1, F3, B4-G4, B5, D5, E5, C6, F6, A9, B9, F9, B10-F10, A12, D12 (24 samples) | Yes |
25 | WRosenthal_Trout_plate25* *Use A7 and B7 from original digest for ligation
| WRT25 |
| C6-H6, H9, Col 10, 11, 12 | 42 samples need dilution - normalize to 140ng/uL | A1, C1, E1, H1, G2, H2, F3-H3, A4, D4, E4, G4, H4, B5, H7, B9, E9 (18 samples) | Yes |
27 | WY_YCT_2021_plate04 | WR_WYCT4 |
| E12, F12 | 87 samples need dilution - normalize to 140ng/uL | B9 | Yes |
28 | WY_YCT_2021_plate09 | WR_WYCT9 |
|
| 77 samples need dilution - normalize to 140ng/uL | F8 | Yes |
29 | WY_YCT_2021_plate10 | WR_WYCT10 |
|
| 91 samples need dilution - normalize to 140ng/uL | C9-F9, H9, A10, C10, F10, G10, C11, D11, F11-H11 (14 samples) | Yes |
30 | WY_YCT_2021_plate11 | WR_WYCT11 |
|
| 77 samples need dilution - normalize to 140ng/uL | None | Yes |
31 | WY_YCT_2021_plate13 | WR_WYCT13 |
|
| 40 samples need dilution - normalize to 140ng/uL | None | Yes |
32 | WY_YCT_2021_plate14 | WR_WYCT14 |
| Col 11, 12 | 50 samples need dilution - normalize to 140ng/uL | None | Yes |
Restriction Digestion
(Keep MM and reaction plates on ice)
Set Incubator to 37 C
Add 3 ul Digestion MM to 32 plates using the 8 channel
Reagent | ul/rxn | rxns | ul needed (1.5x) |
10x T4 Buffer | 1.15 | 1385 | 2389.1 |
5M NaCl | 0.12 | 1385 | 249.3 |
1 mg/ml BSA | 0.6 | 1385 | 1246.5 |
H2O | 0.73 | 1385 | 1516.6 |
MseI (enzyme) | 0.12 | 1385 | 249.3 |
EcoR1 (enzyme) | 0.28 | 1385 | 581.7 |
Total | 3 | 1385 | 6232.5 |
Add 6 ul template to each plate with Benchsmart. Cover and seal each plate, vortex, centrifuge and incubate at 37C for 8 hours. Followed by 65C for 1 hour.