We are expecting little to no difference. So, we will proceed with actual template, but the smaller PCR pool. We will compare via qPCR and bioanalyzer.
Restriction Digestion
(Keep MM and reaction plates on ice)
Add 3 ul Digestion MM to 22 plates using the 8 channel
Reagent | ul/rxn | rxns | ul needed |
|---|---|---|---|
10x T4 Buffer | 1.15 | 450 | 517.5 |
5M NaCl | 0.12 | 450 | 54 |
1 mg/ml BSA | 0.6 | 450 | 270 |
H2O | 0.73 | 450 | 328.5 |
MseI (enzyme) | 0.12 | 450 | 54 |
EcoR1 (enzyme) | 0.28 | 450 | 126 |
Total | 3 | 450 | 1350 |
Add 6 ul template to each plate with Benchsmart. Cover and seal each plate, vortex, centrifuge and incubate at 37C for 8 hours. Followed by 62C for 1 hour. Do 1 set on 2 thermocyclers and 1 set in incubator (Somebody will have to return in 8 hours).
Adaptor Ligation
Spin down plates and add 1.4 ul Ligation MM to each well with 8 channel.
Reagent | ul/rxn | rxns | ul needed |
|---|---|---|---|
MseI oligo | 1 | 440 | 440 |
H2O | 0.112 | 440 | 49.28 |
10x T4 Buffer | 0.1 | 440 | 44 |
5M NaCl | 0.01 | 440 | 4.4 |
1 mg/ml BSA | 0.05 | 440 | 22 |
T4 DNA ligase | 0.1675 | 440 | 73.7 |
Total | 1.4 | 440 | 633.38
|
Add 1 ul of EcoR1 Adaptors with 8-channel and Track which template plate gets which MID plate.
Template | EcoR1 MID plate |
|---|---|
2017_ALFALFA_PLATE10 | GBS Plate 5 |
2017_ALFALFA_PLATE11 | GBS Plate 6 |
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 ligated plates matching well to well in sets of 4 with one set of 2 avoiding combining the same MIDs together:
Pool | Plate1 | Plate2 | Plate3 | Plate4 |
|---|---|---|---|---|
6a | 2017_ALFALFA_PLATE10 | 2017_ALFALFA_PLATE11 |
|
|
6b | 2017_ALFALFA_PLATE10 | 2017_ALFALFA_PLATE11 |
Add 16 ul of PCR1 MM to each well of plates
Reagent | ul/rxn | rxns | ul needed |
|---|---|---|---|
H2O | 9.52 | 250 | 2380 |
5x iProof buffer | 4 | 250 | 1000 |
10 mM dNTPs | 0.4 | 250 | 100 |
50 mM MgCl2 | 0.4 | 250 | 100 |
5 uM Illumina Primers | 1.33 | 250 | 332.5 |
iProof TAQ | 0.2 | 250 | 50 |
DMSO | 0.15 | 250 | 37.5 |
total | 16 | 250 | 4000 |
Add 4 ul of restriction/ligation products to each well
Seal, Vortex, Spin down, and then run on Thermocycler program: GBS1:
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
Reagent | ul/rxn | rxns | ul needed |
|---|---|---|---|
5x Iproof buffer | 0.425 | 244 | 106.25 |
10 mM dNTPs | 0.4 | 244 | 100 |
Primers | 1.33 | 244 | 332.5 |
Total | 2.155 | 244 | 538.75 |
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 |