Setup Notes
~6 ~28 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
Send Mix of on campus and Sending Out for Sequencing
5% PhiX spike
...
Normalize plate reader with Buffer AE TE for all plates
Quantify all plates
Normalize first 3 to 10 ng/ul and others to 30 ng/ul
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Reagent | ul/rxn | rxns | ul needed (1.5x) |
10x T4 Buffer | 1.15 | 800600 | 690 |
5M NaCl | 0.12 | 800600 | 72 |
1 mg/ml BSA | 0.6 | 800600 | 360 |
H2O | 0.73 | 800600 | 438 |
MseI (enzyme) | 0.12 | 800600 | 72 |
EcoR1 (enzyme) | 0.28 | 800600 | 168 |
Total | 3 | 800600 | 1800 |
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 | 600 | 800600800 | 400 | |
H2O | 0.112 | 800 | 89.6600 | 67.2 | 44.8 |
10x T4 Buffer | 0.1 | 600 | 80060 | 8040 | |
5M NaCl | 0.01 | 800600 | 6 | 84 | |
1 mg/ml BSA | 0.05 | 600 | 80030 | 4020 | |
T4 DNA ligase | 0.1675 | 800 | 134600 | 100.5 | 67 |
Total | 1.4 | 800600 | 840 | 1120560 |
Add 1 ul of EcoR1 Adaptors with 96-channel and Track which template plate gets which MID plate.
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GTL Plate | EcoR1 MID plate | Pool Plate | Library | ||
|---|---|---|---|---|---|
YCT_library_22_6_3 | 3Trout1 | 1 | 3Trout_Pool1 | 1 | |
Teton_Library_22_3_1 | 3Trout2 | 2 | 3Trout_Pool1 | 1 | |
Teton_Library_Plate_22_3_2 | 3Trout3 | 3 | 3Trout_Pool2and4 | 1 | |
Tetons_Library_Plate_22_5_4 | 3Trout4 | 4 | 3Trout_Pool2 | 2 | |
YCT_Library_22_3_5 | 3Trout5 | 5 | 3Trout_Pool2 | 2 | |
UMT_YCT_RBT_DNA to UWY | 3Trout6 | 6 | 3Trout_Pool2and4 | 2 | |
Norm to | Sequencer | ||||
WCR31 | 1 | 1+2 | 1 | 10 | Next |
WCR32 | 2 | 1+2 | 1 | 10 | Next |
WCR33 | 3 | 3 | 1 | 10 | Next |
WCR34 | 4 | 4+5 | 2 | 30 | Next |
WCR36 | 5 | 4+5 | 2 | 30 | Next |
WCR37 | 6 | 6+7 | 2 | 30 | Next |
WCR38 | 7 | 6+7 | 3 | 30 | Next |
WCR39 | 8 | 8+9 | 3 | 30 | Next |
WCR40 | 1 | 8+9 | 3 | 30 | Next |
WCR41 | 2 | 10+11 | 4 | 10 | Next |
WCR45 | 3 | 10+11 | 4 | 10 | Next |
WCR46 | 4 | 12 | 4 | 10 | Next |
WCR47 | 5 | 13+14 | 6 | 30 | Nov |
WCR48 | 6 | 13+14 | 6 | 30 | Nov |
WCR49 | 7 | 15+16 | 5 | 40 | Nov |
WCR50 | 8 | 15+16 | 5 | 40 | Nov |
WCR51 | 1 | 17+18 | 5 | 40 | Nov |
WCR52 | 2 | 19+20 | 6 | 30 | Nov |
WCR53 | 3 | 17+18 | 5 | 40 | Nov |
WCR54 | 4 | 19+20 | 6 | 30 | Nov |
WCR43 | 5 | 21+22 | 7 | 30 | Nov |
WCR44 | 6 | 21+22 | 7 | 30 | Nov |
WCR55 | 7 | 23+24 | 7 | 30 | Nov |
WCR56 | 8 | 23+24 | 7 | 30 | Nov |
WCR57 | 1 | 25+26 | 8 | 30 | Next |
WCR58 | 2 | 25+26 | 8 | 30 | Next |
WCR59 | 3 | 27+28 | 8 | 30 | Next |
WCR28 | 7 | 27+28 | 9 | 10* | Next |
WCR35 | 8 | 29+30 | 9 | 10* | Next |
WCR42 | 1 | 29+30 | 9 | 10* | Next |
Label reaction plates with MID plate used.
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Reagent | ul/rxn | rxns | ul needed (x1.4) | |
|---|---|---|---|---|
H2O | 9.52 | 350 | 33323094 | 4760 |
5x iProof buffer | 4 | 350 | 1300 | 14002000 |
10 mM dNTPs | 0.4 | 350 | 140130 | 200 |
50 mM MgCl2 | 0.4 | 350 | 130 | 140200 |
5 uM Illumina Primers | 1.33 | 350 | 465432 | 665 |
iProof TAQ | 0.2 | 350 | 7065 | 100 |
DMSO | 0.15 | 350 | 52.549 | 75 |
total | 16 | 350 | 5200 | 56008000 |
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 | 360 | 153 | 106 | 212.5 |
10 mM dNTPs | 0.4 | 360 | 144 | 100 | 200 |
Primers | 1.33 | 360 | 479 | 332 | 665 |
Total | 2.155 | 360 | 776 | 539 |
Then continue with the last four unlisted steps for GBS1 from above:
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Run Final Product on qPCR for check and quant
Result from qPCR check :
qPCR | Sample | RSB |
|---|---|---|
2.87 | ||
load =800 pM
dilute 1 nM PhiX to 500 pM
...