Setup Notes
~6 plates of trout to be prepared via the MseI and EcoRI GBS library prep protocol with some slight modifications:
Pool ~6*96 individuals per library
Cleanup each full pool via ultra purification
Size select for 300-366bp fragments size window via BluePippin or Pippin Prep
Send Out for Sequencing
5% PhiX spike
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Normalize plate reader with Buffer AE for all plates
Quantify all plates
Check for samples above 150 Normalize first 3 to 10 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 and others to 30 ng/ul
Restriction Digestion
(Keep MM and reaction plates on ice)
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Reagent | ul/rxn | rxns | ul needed (1.5x) |
10x T4 Buffer | 1.15 | 800 | 920690 |
5M NaCl | 0.12 | 800 | 9672 |
1 mg/ml BSA | 0.6 | 800 | 480360 |
H2O | 0.73 | 800 | 584438 |
MseI (enzyme) | 0.12 | 800 | 9672 |
EcoR1 (enzyme) | 0.28 | 800 | 224168 |
Total | 3 | 800 | 24001800 |
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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