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 (5 uM working)
- Illpcr1 (5 uM working)
- Pool of above 2 primers (2.5 uM working of each)
Original SOP
Normalize templates
Use plate reader to quantify templates
Normalize to between 20 ng/ul and 150 ng/ul
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. (This step will also need to be done for the 8th EcoRI adaptor if it is available.)
Restriction Digestion
(Keep MM and reaction plates on ice)
Add 3 ul Digestion MM to 22 plates using the Benchsmart
Reagent | ul/rxn | rxns | ul needed |
|---|---|---|---|
10x T4 Buffer | 1.15 | 2400 | 2760 |
5M NaCl | 0.12 | 2400 | 288 |
1 mg/ml BSA | 0.6 | 2400 | 1440 |
H2O | 0.73 | 2400 | 1752 |
MseI (enzyme) | 0.12 | 2400 | 288 |
EcoR1 (enzyme) | 0.28 | 2400 | 672 |
Total | 3 | 2400 | 7200 |
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 | 2400 | 2400 |
H2O | 0.112 | 2400 | 268.8 |
10x T4 Buffer | 0.1 | 2400 | 240 |
5M NaCl | 0.01 | 2400 | 24 |
1 mg/ml BSA | 0.05 | 2400 | 120 |
T4 DNA ligase | 0.1675 | 2400 | 402 |
Total | 1.4 | 2400 | 3454.8 |
Add 1 ul of EcoR1 Adaptors with 8-channel and Track which template plate gets which MID plate.
Template | EcoR1 MID plate |
|---|---|
2017_ALFALFA_PLATE1 | |
2017_ALFALFA_PLATE2 | |
2017_ALFALFA_PLATE3 | |
2017_ALFALFA_PLATE4 | |
2017_ALFALFA_PLATE5 | |
2017_ALFALFA_PLATE6 | |
2017_ALFALFA_PLATE7 | |
2017_ALFALFA_PLATE8 | |
2017_ALFALFA_PLATE9 | |
2017_ALFALFA_PLATE10 | |
2017_ALFALFA_PLATE11 | |
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 plates matching well to well in sets of 3 or 4 avoiding combining the same MIDs together.
Add 16 ul of PCR1 MM to each well of plates
Reagent | ul/rxn | rxns | ul needed |
|---|---|---|---|
H2O | 9.52 | 777 | 7397.05 |
5x iProof buffer | 4 | 777 | 3108 |
10 mM dNTPs | 0.4 | 777 | 310.8 |
50 mM MgCl2 | 0.4 | 777 | 310.8 |
2.5 uM Illumina Primers | 1.33 | 777 | 1033.4 |
iProof TAQ | 0.2 | 777 | 155.4 |
DMSO | 0.15 | 777 | 116.55 |
total | 16 | 777 | 12432 |
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 | 777 | 330.225 |
10 mM dNTPs | 0.4 | 777 | 310.8 |
Primers | 1.33 | 777 | 1033.41 |
Total | 2.155 | 777 | 1674.435 |
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 |
Size Selection
Blue Pippin?
Gel Extraction?