Setup Notes
...
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 (x1.4) | ||
|---|---|---|---|---|---|
H2O | 9.52 | 576 | 7712 | 5483.52 | 36661904 |
5x iProof buffer | 4 | 576 | 3240 | 2304 | 1536800 |
10 mM dNTPs | 0.4 | 576 | 324 | 230.4153.6 | 80 |
50 mM MgCl2 | 0.4 | 576 | 324 | 230.4153.6 | 80 |
5 uM Illumina Primers | 1.33 | 576 | 1077 | 766.08 | 511266 |
iProof TAQ | 0.2 | 576 | 162 | 115.276.8 | 40 |
DMSO | 0.15 | 576 | 121 | 86.457.6 | 30 |
total | 16 | 576 | 12960 | 9216 | 61443200 |
Add 16 ul of PCR1 MM to each well of four new hard shell PCR plates with 8 channel
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Library | qPCR Result (nM) | RSB ul for 2nM | Library ul for 2 nM | RSB ul for load | Library ul for Load | PhiX ul for load | Loading Concentration Used | % Occupied | Samples | Yield Gbp |
|---|---|---|---|---|---|---|---|---|---|---|
1 | 6.5 | 34.6 | 15.4 | 10.4 | 8 | 1.6 | 384 | |||
2 | 4.7 | 28.7 | 21.3 | 10.4 | 8 | 1.6 | 383 | |||
3 | 4.9 | 29.6 | 20.4 | 10.4 | 8 | 1.6 | 348 | |||
4 | 5.3 | 31.1 | 18.9 | 10.4 | 8 | 1.6 | 382 | |||
5 | 6 | 33.3 | 16.7 | 10.4 | 8 | 1.6 | 374 | |||
6 | 5.5 | 31.8 | 18.2 | 10.4 | 8 | 1.6 | 365 | |||
7 | 3.9 | 24.4 | 25.6 | 11.4 | 7 | 1.6 | 700pM | 336 | ||
8 | 7.6 | 36.8 | 13.2 | 10.4 | 8 | 1.6 | 800 pM | 98.97 | 297 | 126.52 |
1SMIN | 900 | 99.23 | 134.84 |