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 10 uM working)
- Illpcr1 (5 10 uM working)
- Pool of above 2 primers (2.5 uM working of each)
Original SOP
...
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 |
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.
...
Pool plates matching well to well in sets of 4 with one set of 2 avoiding combining the same MIDs together.
...
Reagent | ul/rxn | rxns | ul needed |
|---|---|---|---|
H2O | 9.52 | 650700 | 61886664 |
5x iProof buffer | 4 | 650700 | 26002800 |
10 mM dNTPs | 0.4 | 650700 | 260280 |
50 mM MgCl2 | 0.4 | 650700 | 260280 |
2.5 uM Illumina Primers | 1.33 | 650700 | 864.5931 |
iProof TAQ | 0.2 | 650700 | 130140 |
DMSO | 0.15 | 650700 | 97.5105 |
total | 16 | 650700 | 10400 |
Add 4 ul of restriction/ligation products to each well
...
Reagent | ul/rxn | rxns | ul needed |
|---|---|---|---|
5x Iproof buffer | 0.425 | 650700 | 276297.255 |
10 mM dNTPs | 0.4 | 650700 | 260280 |
Primers | 1.33 | 650700 | 864.5931 |
Total | 2.155 | 650700 | 14001508.755 |
Then continue with the last four unlisted steps for GBS1 from above:
...