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 (2.5 uM working)
- Illpcr1 (2.5 uM working)
Original SOP
Normalize templates
Use plate reader to quantify templates
Normalize to between 20 ng/ul and 150 ng/ul
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 | 1536 (or 768 if not duplicating) | 14622.72 |
5x KAPA HIFI buffer | 4 | 1536 | 6144 |
10 mM dNTPs | 0.4 | 1536 | 614.4 |
50 mM MgCl2 | 0.4 | 1536 | 614.4 |
2.5 uM Illumina Primers | 1.33 | 1536 | 2042.88 |
KAPA HIFI TAQ | 0.2 | 1536 | 307.2 |
DMSO | 0.15 | 1536 | 230.4 |
total | 16 | 1536 | 24806.4 |
Add 4 ul of restriction/ligation products to each well
Extra PCR?
Reagent | ul/rxn | rxns | ul needed |
|---|---|---|---|
5x Iproof buffer | 0.425 | ||
10 mM dNTPs | 0.4 | ||
Primers | 1.33 |