Setup Notes
~6 ~28 plates of trout to be prepared via the MseI and EcoRI GBS library prep protocol with some slight modifications:
...
Reagent | ul/rxn | rxns | ul needed (1.5x) | ||
10x T4 Buffer | 1.15 | 800600 | 690 | 230 | 115 |
5M NaCl | 0.12 | 800600 | 72 | 24 | 12 |
1 mg/ml BSA | 0.6 | 800600 | 360 | 120 | 60 |
H2O | 0.73 | 800600 | 438 | 146 | 73 |
MseI (enzyme) | 0.12 | 800600 | 72 | 24 | 12 |
EcoR1 (enzyme) | 0.28 | 800600 | 168 | 56 | 28 |
Total | 3 | 800600 | 1800 | 600 | 300 |
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 | 600800 | 67.2 | 8944.68 |
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 | 600800 | 100.5 | 13467 |
Total | 1.4 | 600 | 800840 | 1120560 |
Add 1 ul of EcoR1 Adaptors with 96-channel and Track which template plate gets which MID plate.
Template | GTL Plate | EcoR1 MID plate | Pool Plate | Library | |
|---|---|---|---|---|---|
YCT_library_22_6_3 | 3Trout1WCR31 | 13Trout_Pool1 | 1Teton_Library_22_3_+2 | 1 | |
3Trout2WCR32 | 23Trout_Pool1 | 1+2 | 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 | |
WCR33 | 3 | 3 | 1 | ||
WCR34 | 4 | 4+5 | 2 | ||
WCR36 | 5 | 4+5 | 2 | ||
WCR37 | 6 | 6+7 | 2 | ||
WCR38 | 7 | 6+7 | 3 | ||
WCR39 | 8 | 8 | 3 | ||
Label reaction plates with MID plate used.
...
Reagent | ul/rxn | rxns | ul needed (x1.4) | |
|---|---|---|---|---|
H2O | 9.52 | 350 | 3094 | 33321904 |
5x iProof buffer | 4 | 350 | 1300 | 1400800 |
10 mM dNTPs | 0.4 | 350 | 130 | 14080 |
50 mM MgCl2 | 0.4 | 350 | 140130 | 80 |
5 uM Illumina Primers | 1.33 | 350 | 432 | 465266 |
iProof TAQ | 0.2 | 350 | 65 | 7040 |
DMSO | 0.15 | 350 | 49 | 52.530 |
total | 16 | 350 | 5200 | 56003200 |
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 | 360 | 153 | 135.1 |
10 mM dNTPs | 0.4 | 360 | 144 | 127.2 |
Primers | 1.33 | 360 | 479 | 423 |
Total | 2.155 | 360 | 776 |
Then continue with the last four unlisted steps for GBS1 from above:
...