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
Library 1
SAR_Plate1_Lib1
SAR_Plate3_Lib1
SAR_Plate5_Lib1
SAR_Plate7_Lib1
SAR_Plate9_Lib1
SAR_Plate11_Lib1
SAR_Under20_Lib1
Library 2
SAR_Plate2_Lib2
SAR_Plate4_Lib2
SAR_Plate6_Lib2
SAR_Plate8_Lib2
SAR_Plate10_Lib2
SAR_Plate12_Lib2
SAR_Over750_Lib2
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
Plate 12 was diluted 4x by adding 60 ul TE; plate 3x by adding 40ul
Both plates and tubes were requantified
Normalize plates with TE on Nimbus
Restriction Digestion
(Keep MM and reaction plates on ice)
...
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.
...
Reagent | ul/rxn | rxns | ul needed (1.6x) | |
|---|---|---|---|---|
MseI oligo | 1 | 6001600600 | 1600 | 400220 |
H2O | 0.112 | 6001600 | 67179.2 | 4424.86 |
10x T4 Buffer | 0.1 | 6001600 | 60160 | 4022 |
5M NaCl | 0.01 | 6001600 | 6164 | 2.2 |
1 mg/ml BSA | 0.05 | 6001600 | 3080 | 2011 |
T4 DNA ligase | 0.1675 | 6001600 | 100.5268 | 6737 |
Total | 1.4 | 6001600 | 8402240 | 560220 |
Add 1 ul of EcoR1 Adaptors with 96-channel and Track which template plate gets which MID plate.
GTL Plate | EcoR1 MID plate | Pool Plate | Library | Norm to | Sequencer |
|---|---|---|---|---|---|
1Sauger1 | 1 | ||||
1Sauger3 | 2 | ||||
1Sauger5 | 3 | ||||
1Sauger7 | 4 | ||||
1Sauger9 | 5 | ||||
1Sauger11 | 6 | ||||
1Sauger2 | 1 | ||||
1Sauger4 | 2 | ||||
1Sauger6 | 3 | ||||
1Sauger8 | 4 | ||||
1Sauger10 | 5 | ||||
1Sauger12 | 6 | ||||
1SaugerUnderOver | 8 | ||||
Label reaction plates with MID plate used.
...
Reagent | ul/rxn | rxns | ul needed (x1.4) | |
|---|---|---|---|---|
H2O | 9.52 | 350800 | 30947616 | 4760 |
5x iProof buffer | 4 | 350800 | 13003200 | 2000 |
10 mM dNTPs | 0.4 | 350800 | 130 | 200320 |
50 mM MgCl2 | 0.4 | 350800 | 130 | 200320 |
5 uM Illumina Primers | 1.33 | 350800 | 4321064 | 665 |
iProof TAQ | 0.2 | 350800 | 65160 | 100 |
DMSO | 0.15 | 350800 | 49 | 75120 |
total | 16 | 350800 | 5200 | 800012800 |
Add 16 ul of PCR1 MM to each well of four new hard shell PCR plates with 8 channel
...
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 | 360 | 144 | 100 | 200800 | 320 |
Primers | 1.33 | 360800 | 479 | 3321064 | 665 | |
Total | 2.155 | 360800 | 776539 |
Then continue with the last four unlisted steps for GBS1 from above:
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Run Final Products on Tapestation for size check
1Sauger1
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1Sauger2
...