Setup Notes
~18 12 plates plus 32 tubes of Sauger to be prepared via the MseI and EcoRI GBS library prep protocol with some slight modifications:
Cleanup each full pool via ultra purification
Size select for 250-350bp fragments size window via Pippin Prep
Mix of on campus and Sending Out for Sequencing to Admera
5% PhiX spike
Check In Samples Against List from Sam Patrick Johnson
Load Submission Data into MISO
Quantify and normalize samples:
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Normalize plate reader with TE for all plates
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Normalize samples and quantify some:
Dilute Super high tubes an additional 3x by adding 60 ul TE
High plate was diluted 4x by adding 60 ul TE
Normalize plates with TE on Nimbus
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 | 600800 | 690920 |
5M NaCl | 0.12 | 600800 | 7296 |
1 mg/ml BSA | 0.6 | 600800 | 360480 |
H2O | 0.73 | 600800 | 438584 |
MseI (enzyme) | 0.12 | 600800 | 7296 |
EcoR1 (enzyme) | 0.28 | 600800 | 168224 |
Total | 3 | 600800 | 18002400 |
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 | 6001600600 | 1600 | 400 |
H2O | 0.112 | 6001600 | 67179.2 | 44.8 |
10x T4 Buffer | 0.1 | 6001600 | 60 | 40160 |
5M NaCl | 0.01 | 6001600 | 6 | 416 |
1 mg/ml BSA | 0.05 | 6001600 | 30 | 2080 |
T4 DNA ligase | 0.1675 | 6001600 | 100.5268 | 67 |
Total | 1.4 | 6001600 | 840 | 5602240 |
Add 1 ul of EcoR1 Adaptors with 96-channel and Track which template plate gets which MID plate.
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Reagent | ul/rxn | rxns | ul needed (x1.4) | |
|---|---|---|---|---|
H2O | 9.52 | 350800 | 3094 | 47607616 |
5x iProof buffer | 4 | 350800 | 1300 | 20003200 |
10 mM dNTPs | 0.4 | 350800 | 130 | 200320 |
50 mM MgCl2 | 0.4 | 350800 | 130 | 200320 |
5 uM Illumina Primers | 1.33 | 350800 | 432 | 6651064 |
iProof TAQ | 0.2 | 350800 | 65160 | 100 |
DMSO | 0.15 | 350800 | 49 | 75120 |
total | 16 | 350800 | 520012800 | 8000 |
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 | 360800 | 153 | 106 | 212.5 | 340 |
10 mM dNTPs | 0.4 | 360800 | 144 | 100320 | 200 | |
Primers | 1.33 | 360 | 479 | 332 | 665800 | 1064 |
Total | 2.155 | 360800 | 776539 |
Then continue with the last four unlisted steps for GBS1 from above:
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