1BAT
118 samples expected. We will add 2 lambda bacteriophage sample positive controls bto work better with our robots. 124 DNA samples provided. We will add 2 lambda and 2 negative control for 128 total. We will normalize to 8 ng/ul to minimize low samples.
Restriction Digestion
(Keep MM and reaction plates on ice)
Set Incubator to 37 C
Add 3 ul Digestion MM to wells using the 8 channel
Reagent | ul/rxn | rxns | ul needed (1.5x) |
10x T4 Buffer | 1.15 | 180 | 207 |
5M NaCl | 0.12 | 180 | 21.6 |
1 mg/ml BSA | 0.6 | 180 | 108 |
H2O | 0.73 | 180 | 131.4 |
MseI (enzyme) | 0.12 | 180 | 21.6 |
EcoR1 (enzyme) | 0.28 | 180 | 50.4 |
Total | 3 | 180 | 540 |
Add 6 ul template to each plate with Integra Assist Plus. Cover and seal each plate, vortex, centrifuge and incubate at 37C for 8 hours. Followed by 65C for 1 hour on thermocycler.
Adaptor Ligation
Spin down plates and add 1.4 ul Ligation MM to each well with 8 channel.
Reagent | ul/rxn | rxns | ul needed (1.5x) |
|---|---|---|---|
MseI oligo | 1 | 180 | 180 |
H2O | 0.112 | 180 | 20.2 |
10x T4 Buffer | 0.1 | 180 | 18 |
5M NaCl | 0.01 | 180 | 1.8 |
1 mg/ml BSA | 0.05 | 180 | 9 |
T4 DNA ligase | 0.1675 | 180 | 30.2 |
Total | 1.4 | 180 | 252 |
Add 1 ul of EcoR1 Adaptors with 96-channel and track which template plate gets which MID plate. Leave out at room temperature for ~2 hours.
Plate 1 got Adapter Plate 8 and Plate 2 got Adapter plate 7
PCR Amplification
Reagent | ul/rxn | rxns | ul needed (x1.4) |
|---|---|---|---|
H2O | 9.52 | 75 | 857 |
5x iProof buffer | 4 | 75 | 360 |
10 mM dNTPs | 0.4 | 75 | 36 |
50 mM MgCl2 | 0.4 | 75 | 36 |
5 uM Illumina Primers | 1.33 | 75 | 120 |
iProof TAQ | 0.2 | 75 | 18 |
DMSO | 0.15 | 75 | 13.5 |
total | 16 | 75 | 1440 |
Add 16 ul of PCR1 MM to each well.
Add 4uL of template from pools.
Seal, Vortex, Spin down, and then run on Thermocycler program: GBS2:
Temp C | Cycles | Time |
|---|---|---|
95* | 1X | 3:00 |
98 | 20X | 0:30 |
60 | 20X | 0:30 |
72 | 20X | 0:40 |
72 | 1X | 10:00 |
4* | 1X* | 0:00* |
TapeStation and qPCR
For size selection, we ran a tight run targeting 362 bp:
