Setup Notes
~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
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
Quantify and normalize samples:
Normalize plate reader with TE for all plates
Quantify all plates
Normalize first 3 to 10 ng/ul and others to 30 ng/ul
Restriction Digestion
(Keep MM and reaction plates on ice)
Set Incubator to 37 C
Add 3 ul Digestion MM to 32 plates using the 8 channel
Reagent | ul/rxn | rxns | ul needed (1.5x) | |
10x T4 Buffer | 1.15 | 600 | 690 | 437 |
5M NaCl | 0.12 | 600 | 72 | 45.6 |
1 mg/ml BSA | 0.6 | 600 | 360 | 228 |
H2O | 0.73 | 600 | 438 | 277.4 |
MseI (enzyme) | 0.12 | 600 | 72 | 45.6 |
EcoR1 (enzyme) | 0.28 | 600 | 168 | 106.4 |
Total | 3 | 600 | 1800 | 1140 |
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.
Reagent | ul/rxn | rxns | ul needed (1.6x) | |
|---|---|---|---|---|
MseI oligo | 1 | 600 | 600 | 400 |
H2O | 0.112 | 600 | 67.2 | 44.8 |
10x T4 Buffer | 0.1 | 600 | 60 | 40 |
5M NaCl | 0.01 | 600 | 6 | 4 |
1 mg/ml BSA | 0.05 | 600 | 30 | 20 |
T4 DNA ligase | 0.1675 | 600 | 100.5 | 67 |
Total | 1.4 | 600 | 840 | 560 |
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 | 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 | 8 | 30 | Next | ||
WCR28 | 4 | 9 | 10* | Next | ||
WCR35 | 5 | 9 | 10* | Next | ||
WCR42 | 6 | 9 | 10* | Next |
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.
PCR Amplification
PCR1:
Reagent | ul/rxn | rxns | ul needed (x1.4) | |
|---|---|---|---|---|
H2O | 9.52 | 350 | 3094 | 1332.8 |
5x iProof buffer | 4 | 350 | 1300 | 560 |
10 mM dNTPs | 0.4 | 350 | 130 | 56 |
50 mM MgCl2 | 0.4 | 350 | 130 | 56 |
5 uM Illumina Primers | 1.33 | 350 | 432 | 186.2 |
iProof TAQ | 0.2 | 350 | 65 | 28 |
DMSO | 0.15 | 350 | 49 | 21 |
total | 16 | 350 | 5200 | 2240 |
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 | 153 | 65.5 |
10 mM dNTPs | 0.4 | 360 | 144 | 61.6 |
Primers | 1.33 | 360 | 479 | 204.8 |
Total | 2.155 | 360 | 776 | 331.87 |
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):
Run Final Product on qPCR for check and quant
Result from qPCR check:
load =800 pM
dilute 1 nM PhiX to 500 pM