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)
...
EcoRI plate 4 oligo Annealing
SpeedVac plates Stock Plate 7 and Stock Plate 8 to dry oligos
Resuspend plate 4a and 4b to 25 uM. We will have to calculate based off initial expected volume and concentration.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.
...
(Keep MM and reaction plates on ice)
Add 3 ul Digestion MM to 22 20 plates using the Benchsmart
Reagent | ul/rxn | rxns | ul needed |
|---|---|---|---|
10x T4 Buffer | 1.15 | 24002220 | 27602553 |
5M NaCl | 0.12 | 24002220288 | 266.4 |
1 mg/ml BSA | 0.6 | 24002220 | 14401332 |
H2O | 0.73 | 240022201752 | 1620.6 |
MseI (enzyme) | 0.12 | 24002220288 | 266.4 |
EcoR1 (enzyme) | 0.28 | 24002220672 | 621.6 |
Total | 3 | 24002220 | 72006660 |
Use an 8 channel to divvy up 71 ul across a plate and then the Benchsmart to add 3 ul to 22 plates.
...
Reagent | ul/rxn | rxns | ul needed |
|---|---|---|---|
MseI oligo | 1 | 240022502400 | 2250 |
H2O | 0.112 | 24002250 | 268.8252 |
10x T4 Buffer | 0.1 | 24002250 | 240225 |
5M NaCl | 0.01 | 2400225024 | 22.5 |
1 mg/ml BSA | 0.05 | 24002250120 | 112.5 |
T4 DNA ligase | 0.1675 | 24002250402 | 376.875 |
Total | 1.4 | 24002250 | 34543238.8875 |
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 | 1 | ||
2017_ALFALFA_PLATE2 | 2 | ||
2017_ALFALFA_PLATE3 | 3 | ||
2017_ALFALFA_PLATE4 | 4 | ||
2017_ALFALFA_PLATE5 | 5 | ||
2017_ALFALFA_PLATE6 | 6 | ||
2017_ALFALFA_PLATE7 | 7 | ||
2017_ALFALFA_PLATE8 | 8 | ||
2017_ALFALFA_PLATE9 | 2017_ALFALFA_PLATE10 | 2017_ALFALFA_PLATE11 | 1 |
Label reaction plates with MID plate used.
...
Pool | Plate1 | Plate2 | Plate3 | Plate4 |
|---|---|---|---|---|
1 | ||||
2 | ||||
3 | ||||
4 | ||||
56 | ||||
|
|
| ||
|
|
|
Add 16 ul of PCR1 MM to each well of plates
Reagent | ul/rxn | rxns | ul needed |
|---|---|---|---|
H2O | 9.52 | 7001100 | 666410472 |
5x iProof buffer | 4 | 7001100 | 28004400 |
10 mM dNTPs | 0.4 | 7001100 | 280440 |
50 mM MgCl2 | 0.4 | 7001100 | 280440 |
5 uM Illumina Primers | 1.33 | 7001100 | 9311463 |
iProof TAQ | 0.2 | 7001100 | 140220 |
DMSO | 0.15 | 7001100 | 105165 |
total | 16 | 7001100 | 1040017600 |
Add 4 ul of restriction/ligation products to each well
...
Reagent | ul/rxn | rxns | ul needed |
|---|---|---|---|
5x Iproof buffer | 0.425 | 7001100 | 297467.5 |
10 mM dNTPs | 0.4 | 7001100 | 280440 |
Primers | 1.33 | 7001100 | 9311463 |
Total | 2.155 | 7001100 | 15082370.5 |
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
Blue Pippin?
Gel Extraction?We will utilize the Pippin Prep machine on loan from the Ernest Lab.