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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Spin down plates and add 1.4 ul Ligation MM to each well with 8 channel.
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.
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