Setup Notes
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Normalize plate reader with Buffer AE for all plates
Quantify all plates
Check for samples above 150 ng/ul
Transfer 20 ul from all wells of a plate needing normalization to new VWR plate
Add 20 ul TE to all wells above 150 ng/ul
Restriction Digestion
(Keep MM and reaction plates on ice)
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Reagent | ul/rxn | rxns | ul needed (1.6x) |
---|---|---|---|
MseI oligo | 1 | 2000 | 2000 |
H2O | 0.112 | 2000 | 224 |
10x T4 Buffer | 0.1 | 2000 | 200 |
5M NaCl | 0.01 | 2000 | 20 |
1 mg/ml BSA | 0.05 | 2000 | 100 |
T4 DNA ligase | 0.1675 | 2000 | 335 |
Total | 1.4 | 2000 | 2800 |
Reagent | ul/rxn | rxns | ul needed (1.6x) |
---|---|---|---|
MseI oligo | 1 | 3072 | 4915.2 |
H2O | 0.112 | 3072 | 550.5 |
10x T4 Buffer | 0.1 | 3072 | 491.5 |
5M NaCl | 0.01 | 3072 | 49.2 |
1 mg/ml BSA | 0.05 | 3072 | 245.8 |
T4 DNA ligase | 0.1675 | 3072 | 823.3 |
Total | 1.4 | 3072 | 7075.5 |
Add 1 ul of EcoR1 Adaptors with 96-channel and Track which template plate gets which MID plate.
Template | EcoR1 MID plate | Pool Plate | Library | |||
---|---|---|---|---|---|---|
WRosenthal_Trout_plate9plate12 | 1 | 2Trout_Pool1-4 | 12TroutRedoPool1 | 1and2 | ||
WRosenthal_Trout_plate10plate16 | 2 | 2Trout_Pool1-4 | 12TroutRedoPool1 | 1and2 | ||
WRosenthal_Trout_plate11plate18 | 3 | 2Trout_Pool5-8 | 1 | |||
WRosenthal_Trout_plate12 | 4 | 2Trout_Pool5-8 | 1 | |||
WRosenthal_Trout_plate13 | 5 | 2Trout_Pool9-12 | 2 | |||
WRosenthal_Trout_plate14 | 6 | 2Trout_Pool9-12 | 2 | |||
WRosenthal_Trout_plate15 | 7 | 2Trout_Pool13-16 | 2 | |||
WRosenthal_Trout_plate16 | 8 | 2Trout_Pool13-16 | 2 | |||
WRosenthal_Trout_plate17 | 1 | 2Trout_Pool21-24 | 4 | |||
WRosenthal_Trout_plate18 | 2 | 2Trout_Pool21-24 | 4 | |||
WRosenthal_Trout_plate19 | 3 | 2Trout_Pool25-28 | 4 | |||
WRosenthal_Trout_plate20 | 4 | 2Trout_Pool25-28 | 4 | |||
WRosenthal_Trout_plate21 | 5 | 2Trout_Pool17-20 | 3 | |||
WRosenthal_Trout_plate22 | 6 | 2Trout_Pool17-20 | 3 | |||
WRosenthal_Trout_plate24 | 7 | 2Trout_Pool53-56 | 3 | |||
WRosenthal_Trout_plate25 | 8 | 2Trout_Pool53-56 | 3 | |||
MT_YCT_RangewideRADs_plate01 | 1 | 2Trout_Pool29-32 | 52TroutRedoPool2 | 1and2 | ||
Wrosenthal_PM_YCT_plate2 | 4 | 2TroutRedoPool2 | 1and2 | |||
MT_YCT_RangewideRADs_plate01 | 5 | 2TroutRedoPool3 | 3and4 | |||
Wrosenthal_GY21_YCT_tray6 | 6 | 2TroutRedoPool3 | 3and4 | |||
WY_YCT_2021_plate14 | 7 | 2TroutRedoPool4 | 3and4 | |||
Wrosenthal_PM_YCT_plate1 | 8 | 2TroutRedoPool4 | 3and4 | |||
WRosenthal_Trout_plate9 | 1 | 2TroutRedoPool5 | 5and6 | |||
WRosenthal_Trout_plate14 | 2 | 2TroutRedoPool5 | 5and6 | |||
WRosenthal_Trout_plate20 | 3 | 2TroutRedoPool6 | 5and6 | |||
WRosenthal_Trout_plate21 | 4 | 2TroutRedoPool6 | 5and6 | |||
WY_YCT_2021_plate10 | 5 | 2TroutRedoPool7 | 7and8 | |||
WY_YCT_2021_plate13 | 6 | 2TroutRedoPool7 | 7and8 | |||
WRosenthal_Trout_plate10 | 1 | 2TroutRedoPool8 | 7and8 | |||
WRosenthal_Trout_plate13 | 2 | 2TroutRedoPool8 | 7and8 | |||
WRosenthal_Trout_plate11 | 1 | 2TroutRedoPool9 | 10and14 | |||
MT_YCT_RangewideRADs_plate03 | 2 | 2Trout_Pool29-32 | 5 | |||
WY_YCT_2021_plate04 | 3 | 2Trout_Pool33-36 | 5 | |||
2TroutRedoPool9 | 9and12 | |||||
WY_YCT_2021_plate09plate11 | 4 | 2Trout_Pool33-36 | 5 | |||
WY_YCT_2021_plate10 | 5 | 2Trout_Pool37-40 | 6 | |||
WY_YCT_2021_plate11 | 6 | 2Trout_Pool37-40 | 66 | 2TroutRedoPool9 | 9and12 | |
MT_WCT_plate15 | ||||||
WRosenthal_Trout_plate19 | 3 | 2TroutRedoPool10 | 10and14 | |||
WY_YCT_2021_plate13plate04 | 7 | 2Trout_Pool49-52 | 62TroutRedoPool11 | 11and13 | ||
WY_YCT_2021_plate14plate09 | 8 | 2Trout_Pool49-52 | 65 | 2TroutRedoPool11 | 11and13 | |
Wrosenthal_GY21_YCT_tray1tray3 | 4 | 2Trout_Pool45-48 | 87 | 2TroutRedoPool12 | 9and12 | |
Wrosenthal_GY21_YCT_tray3tray7 | 5 | 2Trout_Pool45-48 | 8 | |||
Wrosenthal_GY21_YCT_tray4 | 6 | 2Trout_Pool61-64 | 8 | |||
Wrosenthal_GY21_YCT_tray6 | 7 | 2Trout_Pool57-60 | 7 | |||
Wrosenthal_PM_YCT_plate2 | 3 | 2Trout_Pool41-44 | 7 | |||
Wrosenthal_PM_YCT_plate1 | 2 | 2Trout_Pool41-44 | 7 | |||
MT_WCT_plate15 | 1 | 2Trout_Pool61-64 | 88 | 2TroutRedoPool12 | 9and12 | |
WRosenthal_Trout_plate17 | 3 | 2TroutRedoPool13 | 11and13 | |||
WRosenthal_Trout_plate24 | 4 | 2TroutRedoPool13 | 11and13 | |||
WRosenthal_Trout_plate15 | 2 | 2TroutRedoPool14 | 10and14 | |||
WRosenthal_Trout_plate22 | 4 | 2TroutRedoPool14 | 10and14 | |||
WRosenthal_Trout_plate25 | 8 | 2TroutRedoPool15 | 15and16 | |||
Wrosenthal_GY21_YCT_tray7 | 8 | 2Trout_Pool57-60 | 7tray4 | 6 | 2TroutRedoPool15 | 15and16 |
Wrosenthal_GY21_YCT_tray1 | 7 | 2TroutRedoPool16 | 15and16 | |||
Label reaction plates with MID plate used.
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Reagent | ul/rxn | rxns | ul needed (x1.4) | ||
---|---|---|---|---|---|
H2O | 9.52 | 576 | 7712 | 5483.52 | 19045712 |
5x iProof buffer | 4 | 576 | 3240 | 23042400 | 800 |
10 mM dNTPs | 0.4 | 576 | 324 | 230.4240 | 80 |
50 mM MgCl2 | 0.4 | 576 | 324 | 230.4240 | 80 |
5 uM Illumina Primers | 1.33 | 576 | 1077 | 766.08798 | 266 |
iProof TAQ | 0.2 | 576 | 162 | 115.2120 | 40 |
DMSO | 0.15 | 576 | 121 | 86.490 | 30 |
total | 16 | 576 | 12960 | 9216 | 32009600 |
Add 16 ul of PCR1 MM to each well of four new hard shell PCR plates with 8 channel
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Reagent | ul/rxn | rxns | ul needed (x 1.6) | |
5x Iproof buffer | 0.425 | 1152 | 784 | 163.2278 |
10 mM dNTPs | 0.4 | 1152 | 738 | 153.6262 |
Primers | 1.33 | 1152 | 2452 | 511869 |
Total | 2.155 | 1152 | 3973.82828 |
Then continue with the last four unlisted steps for GBS1 from above:
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