Wagner Trout 5 (5Trout)
Setup Notes
9 plates of trout 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 300-366bp fragments size window via Pippin Prep
Mix of on campus and Sending Out for Sequencing
5% PhiX spike
Check In Samples Against List from Will Rosenthal
Load Submission Data into MISO
Pooling Notes
Library 1: MPR1; MPR2; YCT6
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Library 2: MPR3; MPR5; GYCT3
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Library 3: MPR4; MPR6; GYCT7
Quantify and normalize samples:
Normalize plate reader with TE for all plates
Quantify all plates
Normalize to 30 ng/ul
Restriction Digestion
(Keep MM and reaction plates on ice)
Set Incubator to 37 C
Add 3 ul Digestion MM to 9 plates using the 8 channel
Reagent | ul/rxn | rxns | ul needed (1.5x) |
10x T4 Buffer | 1.15 | 860 | 989 |
5M NaCl | 0.12 | 860 | 103.2 |
1 mg/ml BSA | 0.6 | 860 | 516 |
H2O | 0.73 | 860 | 628 |
MseI (enzyme) | 0.12 | 860 | 103.2 |
EcoR1 (enzyme) | 0.28 | 860 | 241 |
Total | 3 | 860 | 2580 |
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.
Adaptor Ligation
Spin down plates and add 1.4 ul Ligation MM to each well with 8 channel of the 7 new plates and the plates from 1BROSE.
Reagent | ul/rxn | rxns | ul needed (1.6x) |
---|---|---|---|
MseI oligo | 1 | 1400 | 1400 |
H2O | 0.112 | 1400 | 156.8 |
10x T4 Buffer | 0.1 | 1400 | 140 |
5M NaCl | 0.01 | 1400 | 14 |
1 mg/ml BSA | 0.05 | 1400 | 70 |
T4 DNA ligase | 0.1675 | 1400 | 234.5 |
Total | 1.4 | 1400 | 1960 |
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 |
---|---|---|---|---|---|
MPR1 | 1 | Â | 1 | 30 | Next |
MPR2 | 2 | Â | 1 | 30 | Next |
YCT6 | 3 | Â | 1 | 30 | Next |
MPR3 | 1 | Â | 2 | 30 | Next |
MPR5 | 2 | Â | 2 | 30 | Next |
GYTC3 | 7 or 8 | Â | 2 | 30 | Next |
MPR4 | 1 | Â | 3 | 30 | Next |
MPR6 | 2 | Â | 3 | 30 | Next |
GYTC7 | 8 or 7 | Â | 3 | 30 | Next |
Label reaction plates with MID plate used.
Cover, vortex, and spin plates. Incubate plates at RT for 2 hours on benchtop.
Add 68 ul of low EDTA TE store at 4C for a month or -20C for longer.
PCR Amplification
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 PCR1:
Reagent | ul/rxn | rxns | ul needed (x1.4) | Â |
---|---|---|---|---|
H2O | 9.52 | 350 | 3094 | 4760 |
5x iProof buffer | 4 | 350 | 1300 | 2000 |
10 mM dNTPs | 0.4 | 350 | 130 | 200 |
50 mM MgCl2 | 0.4 | 350 | 130 | 200 |
5 uM Illumina Primers | 1.33 | 350 | 432 | 665 |
iProof TAQ | 0.2 | 350 | 65 | 100 |
DMSO | 0.15 | 350 | 49 | 75 |
total | 16 | 350 | 5200 | 8000 |
Add 16 ul of PCR1 MM to each well of four new hard shell PCR plates with 8 channel
Add 4uL of template from pooled plates with Benchsmart
Seal, Vortex, Spin down, and then run on Thermocycler program: GBS1:
Temp C | Cycles | Time |
---|---|---|
95* | 1X | 3:00 |
98 | 30X | 0:30 |
60 | 30X | 0:30 |
72 | 30X | 0:40 |
72 | 1X | 10:00 |
4* | 1X* | 0:00* |
*pause step
Extra PCR
Add 2.155 ul of the MM directly to the previous PCR
Reagent | ul/rxn | rxns | ul needed (x 1.6) |
5x Iproof buffer | 0.425 | 360 | 230 |
10 mM dNTPs | 0.4 | 360 | 92 |
Primers | 1.33 | 360 | 306 |
Total | 2.155 | 360 | 496 |
Then continue with the last four unlisted steps for GBS1 from above:
Temp C | Cycles | Time |
---|---|---|
98 | 1X | 3:00 |
60 | 1X | 2:00 |
72 | 1X | 10:00 |
4 | 1X | 0:00 |
Pool Using PCR Plates with Assist Plus
Organize plates by Library # above, vortex, and quick spin
Use 4GbsPoolx8
Use single channel to combine 48 ul from each well of a column into a separate labeled tube.
Pippin Prep Size Select (300-366 bp select):
 First attempt showed too little product. I will concentrate via speedvac and cleanup concentration via Nucleofast 96 PCR.
Run Final Products on Tapestation for size check
Run Final Product on qPCR for check and quant
Result from qPCR check :
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Sample Name | qPCR | Sample ul | RSB ul |
---|---|---|---|
Exp5Trout1 | 1.86 nM | 53.8 | 46.2 |
5Trout1Cleaned | 9.02 nM | 11.1 | 88.9 |
5Trout1Org | 3.05 nM | 32.8 | 67.2 |
load =800 pM
80 ul 1 nM stock, 4 ul 1 nM PhiX, 16 ul RSB