| Info |
|---|
As of 12-08-02-2021: 1000 3700 fish DNA samples expected for Restriction Fragment Sequencing library prep. They will be split into 2 relatively even pools to avoid read count disparity and allow resolution of each sample2-3 plates will be pooled for PCR and sequencing. The first 12 may arrive by 12-13-21. The below tables will need to be adjusted once we settle on how many we are doing at a time. |
Setup Notes
~40 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 | 10003700 | 17256387 |
5M NaCl | 0.12 | 10003700 | 180666 |
1 mg/ml BSA | 0.6 | 10003700 | 9003332 |
H2O | 0.73 | 10003700 | 10954056 |
MseI (enzyme) | 0.12 | 10003700 | 180666 |
EcoR1 (enzyme) | 0.28 | 10003700 | 4201555 |
Total | 3 | 10003700 | 450016662 |
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 | 10003700 | 16005920 |
H2O | 0.112 | 10003700 | 179663.28 |
10x T4 Buffer | 0.1 | 10003700 | 160592 |
5M NaCl | 0.01 | 1000370016 | 59.2 |
1 mg/ml BSA | 0.05 | 10003700 | 80296 |
T4 DNA ligase | 0.1675 | 10003700 | 268991 |
Total | 1.4 | 10003700 | 22408462 |
Add 1 ul of EcoR1 Adaptors with 8-channel and Track which template plate gets which MID plate.
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Pool | Plate7 | Plate8 | Plate9 | Plate10 |
|---|---|---|---|---|
2B |
Pool | Plate11 Col 1-3 | Plate11 Col 4-6 | Plate11 Col 7-9 | Plate11 Col 10-12 |
|---|---|---|---|---|
2C |
Pool | Plate11 Col 1-3 | Plate11 Col 6-4 | Plate11 Col 7-9 | Plate11 Col 12-10 |
|---|---|---|---|---|
2D |
PCR1:
Make MM1 in a 15 ml tube:
Reagent | ul/rxn | rxns | ul needed (x1.4) |
|---|---|---|---|
H2O | 9.52 | 5001850 | 666424658 |
5x iProof buffer | 4 | 5001850 | 280010360 |
10 mM dNTPs | 0.4 | 5001850 | 2801036 |
50 mM MgCl2 | 0.4 | 5001850 | 2801036 |
5 uM Illumina Primers | 1.33 | 5001850 | 9313444 |
iProof TAQ | 0.2 | 5001850 | 140518 |
DMSO | 0.15 | 5001850 | 105388 |
total | 16 | 5001850 | 11,20041440 |
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 | 2401850 | 3401258 |
10 mM dNTPs | 0.4 | 2401850 | 3201184 |
Primers | 1.33 | 24018501064 | 3936.8 |
Total | 2.155 | 24018501724 | 6378.8 |
Then continue with the last four unlisted steps for GBS1 from above:
...