As of 12-08-2021: 3700 fish DNA samples expected for Restriction Fragment Sequencing library prep. 2-3 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. |
~40 plates of trout to be prepared via the MseI and EcoRI GBS library prep protocol with some slight modifications:
Only 2-3 plates will be included in each pool
Pooling of samples for PCR will either only be done in groups of three or handled by half plates in sets of 4
Each Pool will be run by itself on an SP 1x100 NovaSeq
Load Submission Data into MISO
Quantify DNA samples and normalize if any are higher than 150 ng/ul.
(Keep MM and reaction plates on ice)
Set Incubator to 37 C
Add 3 ul Digestion MM to 11 plates using the 8 channel
Reagent | ul/rxn | rxns | ul needed (1.5x) |
10x T4 Buffer | 1.15 | 3700 | 6387 |
5M NaCl | 0.12 | 3700 | 666 |
1 mg/ml BSA | 0.6 | 3700 | 3332 |
H2O | 0.73 | 3700 | 4056 |
MseI (enzyme) | 0.12 | 3700 | 666 |
EcoR1 (enzyme) | 0.28 | 3700 | 1555 |
Total | 3 | 3700 | 16662 |
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.
Spin down plates and add 1.4 ul Ligation MM to each well with 8 channel.
Reagent | ul/rxn | rxns | ul needed (1.6x) |
---|---|---|---|
MseI oligo | 1 | 3700 | 5920 |
H2O | 0.112 | 3700 | 663.8 |
10x T4 Buffer | 0.1 | 3700 | 592 |
5M NaCl | 0.01 | 3700 | 59.2 |
1 mg/ml BSA | 0.05 | 3700 | 296 |
T4 DNA ligase | 0.1675 | 3700 | 991 |
Total | 1.4 | 3700 | 8462 |
Add 1 ul of EcoR1 Adaptors with 8-channel and Track which template plate gets which MID plate.
Template | EcoR1 MID plate | Pool |
---|---|---|
1 | ||
1 | ||
1 | ||
1 | ||
1 | ||
1 | ||
2 | ||
2 | ||
2 | ||
2 | ||
2 |
Label reaction plates with MID plate used.
Cover, vortex, and spin plates. Incubate plates at RT for 2 hours on benchtop.
Add 120 ul of low EDTA TE store at 4C for a month or -20C for longer.
Create working pools. For Pool A combine ligated plates matching well to well. For pool B combine ligated plates with even plates flipped upside down (H12 in A1). C1 and C2 will be recombining Plate 5 column wise. Combine columns 1-3, 4-6, 7-9 and 10-12 into columns 1-3 of a new plate. Then, to columns 7-9 of the same plate, add columns 1-3 and 7-9. Flip Plate 5 around and then pipette 6-4 and 12-10 into columns 7-9 of the pool plate. Mix together 20 ul from each well of each plate for pooled plate using the Benchsmart. After pooling all samples, vortex and spin down plates then use 4 ul from the pooled plates for PCR templates.
Pool | Plate1 | Plate2 | Plate3 | Plate4 |
---|---|---|---|---|
1A |
Pool | Plate1 | Plate2 | Plate3 | Plate4 |
---|---|---|---|---|
1B |
Pool | Plate5 Col 1-6 | Plate5 Col 7-12 | Plate6 Col 1-6 | Plate6 Col 7-12 |
---|---|---|---|---|
1C |
Pool | Plate5 Col 1-6 | Plate5 Col 12-7 | Plate6 Col 1-6 | Plate6 Col 12-7 |
---|---|---|---|---|
1D |
Pool | Plate7 | Plate8 | Plate9 | Plate10 |
---|---|---|---|---|
2A |
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 |
Make MM1 in a 15 ml tube:
Reagent | ul/rxn | rxns | ul needed (x1.4) |
---|---|---|---|
H2O | 9.52 | 1850 | 24658 |
5x iProof buffer | 4 | 1850 | 10360 |
10 mM dNTPs | 0.4 | 1850 | 1036 |
50 mM MgCl2 | 0.4 | 1850 | 1036 |
5 uM Illumina Primers | 1.33 | 1850 | 3444 |
iProof TAQ | 0.2 | 1850 | 518 |
DMSO | 0.15 | 1850 | 388 |
total | 16 | 1850 | 41440 |
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
Add 2.125 ul of the MM directly to the previous PCR
Reagent | ul/rxn | rxns | ul needed (x 1.6) |
5x Iproof buffer | 0.425 | 1850 | 1258 |
10 mM dNTPs | 0.4 | 1850 | 1184 |
Primers | 1.33 | 1850 | 3936.8 |
Total | 2.155 | 1850 | 6378.8 |
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 |
Result from qPCR check: