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
Illumina Primer Pooling
Add 900 ul std TE to 5 tubes
...
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.
...
Heat to 95C for 5 minutes and allow to slowly cool to RT.
EcoRI plate 4 oligo Annealing
SpeedVac plates to dry oligos
...
Heat to 95C for 5 minutes and allow to slowly cool to RT
Restriction Digestion
(Keep MM and reaction plates on ice)
...
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.
...
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:
...
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
...
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?
...