Use iProof Polymerase for this. We can play around with KAPA afterwards to see if we don’t need 2 taqs in house
Reagents and Ordering
- EcoR1 (20,000 units/ml)
- MseI (10,000 units/ml)
- T4 DNA ligase buffer (400,000 units/ml)
- DNA polymerase (BioRad iProof or KAPA HiFi)
- BSA (1 mg/ml)-We have this in 20 mg/ul
- 1 M NaCl-We have this at 5M
- DMSO
- EcoR1 adaptors (We need 1 uM MIDed in plates)
- Mse1 Adaptors (10 uM stock probably need to dilute from 100 uM)
- IIllpcr2 (10 uM working)
- Illpcr1 (10 uM working)
- Pool of above 2 primers (5 uM working of each)
Original SOP
Normalize templates
Use plate reader to quantify templates
Normalize to between 20 ng/ul and 150 ng/ul
2017 alfalfa: Transfer to conical pcr plates. Normalize on nimbus to 100 ng/ul in 30 ul.
Illumina Primer Pooling
Add 900 ul std TE to 5 tubes
Add 50 ul of both Illpcr1 and Illpcr2 to each tube
Seal, vortex, and spin tubes. You will need 2 tubes for this prep.
MseI oligo Annealing
Only needs to be done after first making working stock.
Add 1200 ul std TE to two 2 ml tubes.
Add 150 ul MseI1 to each tube
Add 150 ul Mse2 to each tube
Close, vortex, and spin down both tubes.
Heat to 95C for 5 minutes and allow to slowly cool to RT.
EcoRI plate 4 oligo Annealing
SpeedVac Stock Plate 7 and Stock Plate 8 to dry oligos
Resuspend with 20 ul H2O and 60 ul std TE to avoid over saturating with Tris and EDTA to create 25 uM stocks.
Combine 4 ul from each plate in a well to well fashion with 92 ul of TE to create a 1 uM working plate.
Label it “GBS Working Stock Plate 4” Seal, Vortex, and Spin down resulting plate.
Heat to 95C for 5 minutes and allow to slowly cool to RT
Restriction Digestion
(Keep MM and reaction plates on ice)
Add 3 ul Digestion MM to 16 plates using the Benchsmart
Reagent | ul/rxn | rxns | ul needed |
|---|---|---|---|
10x T4 Buffer | 1.15 | 1800 | 2070 |
5M NaCl | 0.12 | 1800 | 216 |
1 mg/ml BSA | 0.6 | 1800 | 1080 |
H2O | 0.73 | 1800 | 1314 |
MseI (enzyme) | 0.12 | 1800 | 216 |
EcoR1 (enzyme) | 0.28 | 1800 | 504 |
Total | 3 | 1800 | 5400 |
Use an 8 channel to divvy up 71 ul across a plate and then the Benchsmart to add 3 ul to 22 plates.
Add 6 ul template to each plate with 8 channel. Cover and seal each plate, vortex, centrifuge and incubate at 37C for 8 hours. Followed by 62C for 1 hour.
Adaptor Ligation
Spin down plates and add 1.4 ul Ligation MM to each well with Benchsmart.
Reagent | ul/rxn | rxns | ul needed |
|---|---|---|---|
MseI oligo | 1 | 1800 | 1800 |
H2O | 0.112 | 1800 | 201.6 |
10x T4 Buffer | 0.1 | 1800 | 180 |
5M NaCl | 0.01 | 1800 | 18 |
1 mg/ml BSA | 0.05 | 1800 | 90 |
T4 DNA ligase | 0.1675 | 1800 | 301.5 |
Total | 1.4 | 1800 | 2591.1 |
Add 1 ul of EcoR1 Adaptors with 8-channel and Track which template plate gets which MID plate.
Template | EcoR1 MID plate |
|---|---|
2017_ALFALFA_PLATE5 | 5 |
2017_ALFALFA_PLATE6 | 6 |
2017_ALFALFA_PLATE7 | 7 |
2017_ALFALFA_PLATE8 | 8 |
2017_ALFALFA_PLATE9 | 1 |
Label reaction plates with MID plate used.
Cover, vortex, and spin plates. Incubate at 16C for 6 hours.
Add 189 ul of low EDTA TE store at 4C for a month or -20C for longer.
PCR Amplification
Pool ligated plates matching well to well in sets of 4 with one set of 2 avoiding combining the same MIDs together:
Pool | Plate1 | Plate2 | Plate3 | Plate4 |
|---|---|---|---|---|
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2 | ||||
3 | ||||
4 | ||||
5 | ||||
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We are running duplicate PCRs for each template.
Add 16 ul of PCR1 MM to each well of plates
Reagent | ul/rxn | rxns | ul needed |
|---|---|---|---|
H2O | 9.52 | 900 | 8568 |
5x iProof buffer | 4 | 900 | 3600 |
10 mM dNTPs | 0.4 | 900 | 360 |
50 mM MgCl2 | 0.4 | 900 | 360 |
5 uM Illumina Primers | 1.33 | 900 | 1197 |
iProof TAQ | 0.2 | 900 | 180 |
DMSO | 0.15 | 900 | 135 |
total | 16 | 900 | 14400 |
Add 4 ul of restriction/ligation products to each well
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 2.125 ul of the MM directly to the previous PCR
Reagent | ul/rxn | rxns | ul needed |
|---|---|---|---|
5x Iproof buffer | 0.425 | 900 | 382.5 |
10 mM dNTPs | 0.4 | 900 | 360 |
Primers | 1.33 | 900 | 1197 |
Total | 2.155 | 900 | 1939.5 |
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 |
Pooling
Sequencing Pool | PCR Pool | PCR Pool |
|---|---|---|
seqPool1 | Pool1 | Pool2 |
seqPool2 | Pool3 | Pool4 |
seqPool3 | Pool5 | Pool6 |
Size Selection
We will utilize the Pippin Prep machine on loan from the Ernest Lab.