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Normalize plate reader with Buffer AE TE for all plates
Quantify all plates
Normalize first 3 to 10 ng/ul and others to 30 ng/ul
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Reagent | ul/rxn | rxns | ul needed (1.5x) | ||
10x T4 Buffer | 1.15 | 600 | 690 | 230805 | 115 |
5M NaCl | 0.12 | 600 | 72 | 2484 | 12 |
1 mg/ml BSA | 0.6 | 600 | 360 | 120420 | 60 |
H2O | 0.73 | 600 | 438 | 146511 | 73 |
MseI (enzyme) | 0.12 | 600 | 72 | 24 | 1284 |
EcoR1 (enzyme) | 0.28 | 600 | 168 | 56196 | 28 |
Total | 3 | 600 | 1800 | 6002100 | 300 |
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 in EcoBGC oven for 1 hour.
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Reagent | ul/rxn | rxns | ul needed (1.6x) | |
|---|---|---|---|---|
MseI oligo | 1 | 600 | 600 | 400700 |
H2O | 0.112 | 600 | 67.2 | 4478.84 |
10x T4 Buffer | 0.1 | 600 | 60 | 4070 |
5M NaCl | 0.01 | 600 | 6 | 47 |
1 mg/ml BSA | 0.05 | 600 | 30 | 2035 |
T4 DNA ligase | 0.1675 | 600 | 100.5 | 67117.2 |
Total | 1.4 | 600 | 840 | 560980 |
Add 1 ul of EcoR1 Adaptors with 96-channel and Track which template plate gets which MID plate.
Template | GTL Plate | EcoR1 MID plate | Pool Plate | Library | Norm to | Sequencer |
|---|---|---|---|---|---|---|
WCR31 | 1 | 1+2 | 1 | 10 | Next | |
WCR32 | 2 | 1+2 | 1 | 10 | Next | |
WCR33 | 3 | 3 | 1 | 10 | Next | |
WCR34 | 4 | 4+5 | 2 | 30 | Next | |
WCR36 | 5 | 4+5 | 2 | 30 | Next | |
WCR37 | 6 | 6+7 | 2 | 30 | Next | |
WCR38 | 7 | 6+7 | 3 | 30 | Next | |
WCR39 | 8 | 8+9 | 3 | 30 | Next | |
WCR40 | 1 | 8+9 | 3 | 30 | Next | |
WCR41 | 2 | 10+11 | 4 | 10 | Next | |
WCR45 | 3 | 10+11 | 4 | 10 | Next | |
WCR46 | 4 | 12 | 4 | 10 | Next | |
WCR47 | 5 | 13+14 | 6 | 30 | Nov | |
WCR48 | 6 | 13+14 | 6 | 30 | Nov | |
WCR49 | 7 | 15+16 | 5 | 40 | Nov | |
WCR50 | 8 | 15+16 | 5 | 40 | Nov | |
WCR51 | 1 | 17+18 | 5 | 40 | Nov | |
WCR52 | 2 | 19+20 | 6 | 30 | Nov | |
WCR53 | 3 | 17+18 | 5 | 40 | Nov | |
WCR54 | 4 | 19+20 | 6 | 30 | Nov | |
WCR43 | 5 | 7 | 30 | Nov | ||
WCR44 | 6 | 7 | 30 | Nov | ||
WCR55 | 7 | 7 | 30 | Nov | ||
WCR56 | 8 | 7 | 30 | Nov | ||
WCR57 | 1 | 8 | 30 | Next | ||
WCR58 | 2 | 8 | 30 | Next | ||
WCR59 | 3 | 8 | 30 | Next | ||
9 | Next | |||||
9 | Next | |||||
9 | Next | |||||
Label reaction plates with MID plate used.
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