Final RFS Plates using Incubator_Run3
Normalize templates
Use plate reader to quantify templates
Normalize to between 20 ng/ul and 150 ng/ul
2017 alfalfa: Transfer to conical pcr plates. Normalize on nimbus to 100 ng/ul in 30 ul.
Templates:
ALF_PLT5_2017_GBS | ALF_PLT6_2017_GBS | ALF_PLT7_2017_GBS | ALF_PLT8_2017_GBS | ALF_PLT9_2017_GBS |
ALF_PLT1_2020_GBS | ALF_PLT2_2020_GBS | ALF_PLT3_2020_GBS | ALF_PLT4_2020_GBS | ALF_PLT5_2020_GBS |
ALF_PLT6_2020_GBS | ALF_PLT1_2020_END | ALF_PLT2_2020_END | ALF_PLT3_2020_END | ALF_PLT4_2020_END |
ALF_PLT5_2020_END | ALF_PLT6_2020_END | ALF_PLT7_2020_END | ALF_PLT8_2020_END |
|
Restriction Digestion_GBS_Run3_Part1
(Keep MM and reaction plates on ice)
Add 3 ul Digestion MM to 10 plates using the 8 channel
Reagent | ul/rxn | rxns | ul needed |
|---|---|---|---|
10x T4 Buffer | 1.15 | 1050 | 1207.5 |
5M NaCl | 0.12 | 1050 | 126 |
1 mg/ml BSA | 0.6 | 1050 | 630 |
H2O | 0.73 | 1050 | 766.5 |
MseI (enzyme) | 0.12 | 1050 | 126 |
EcoR1 (enzyme) | 0.28 | 1050 | 294 |
Total | 3 | 1050 | 3150 |
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 65C for 1 hour.
Adaptor Ligation_GBS_Run3_Part1
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 | 1075 | 1050 |
H2O | 0.112 | 1075 | 117.6 |
10x T4 Buffer | 0.1 | 1075 | 105 |
5M NaCl | 0.01 | 1075 | 10.5 |
1 mg/ml BSA | 0.05 | 1075 | 52.5 |
T4 DNA ligase | 0.1675 | 1075 | 175.9 |
Total | 1.4 | 1075 | 1511.5 |
Add 1 ul of EcoR1 Adaptors with 8-channel and Track which template plate gets which MID plate.
Template | EcoR1 MID plate |
|---|---|
ALF_PLT5_2017_GBS | EcoR1 Plate7 |
ALF_PLT6_2017_GBS | EcoR1 Plate 8 |
ALF_PLT7_2017_GBS | EcoR1 Plate 1 |
ALF_PLT8_2017_GBS | EcoR1 Plate 2 |
ALF_PLT9_2017_GBS | EcoR1 Plate 3 |
ALF_PLT1_2020_GBS | EcoR1 Plate 4 |
ALF_PLT2_2020_GBS | EcoR1 Plate 5 |
ALF_PLT3_2020_GBS | EcoR1 Plate 6 |
ALF_PLT4_2020_GBS | EcoR1 Plate 7 |
ALF_PLT5_2020_GBS | EcoR1 Plate 8 |
Label reaction plates with MID plate used.
Cover, vortex, and spin plates. Incubate plates at RT for 2 hours on benchtop.
Add 120 ul of low EDTA TE store at 4C for a month or -20C for longer.
Restriction Digestion_GBS_Run3_Part2
(Keep MM and reaction plates on ice)
Add 3 ul Digestion MM to 9 plates using the 8 channel
Reagent | ul/rxn | rxns | ul needed |
|---|---|---|---|
10x T4 Buffer | 1.15 | 950 | 1092.5 |
5M NaCl | 0.12 | 950 | 114 |
1 mg/ml BSA | 0.6 | 950 | 570 |
H2O | 0.73 | 950 | 693.5 |
MseI (enzyme) | 0.12 | 950 | 114 |
EcoR1 (enzyme) | 0.28 | 950 | 266 |
Total | 3 | 950 | 2850 |
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 65C for 1 hour.
Adaptor Ligation_GBS_Run3_Part2
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 | 1050 | 1050 |
H2O | 0.112 | 1050 | 117.6 |
10x T4 Buffer | 0.1 | 1050 | 105 |
5M NaCl | 0.01 | 1050 | 10.5 |
1 mg/ml BSA | 0.05 | 1050 | 52.5 |
T4 DNA ligase | 0.1675 | 1050 | 175.9 |
Total | 1.4 | 1050 | 1470 |
Add 1 ul of EcoR1 Adaptors with 8-channel and Track which template plate gets which MID plate.
Template | EcoR1 MID plate |
|---|---|
ALF_PLT6_2020_GBS | EcoR1 Plate 1 |
ALF_PLT1_2020_END | EcoR1 Plate 2 |
ALF_PLT2_2020_END | EcoR1 Plate 3 |
ALF_PLT3_2020_END | EcoR1 Plate 4 |
ALF_PLT4_2020_END | EcoR1 Plate 5 |
ALF_PLT5_2020_END | EcoR1 Plate 6 |
ALF_PLT6_2020_END | EcoR1 Plate 7 |
ALF_PLT7_2020_END | EcoR1 Plate 8 |
ALF_PLT8_2020_END | EcoR1 Plate 1 |
Label reaction plates with MID plate used.
Cover, vortex, and spin plates. Incubate plates at RT for 2 hours on benchtop.
Add 120 ul of low EDTA TE store at 4C for a month or -20C for longer.
PCR Amplification_GBS_Run3
Create working pools. For pools 3A, 3C, 3E, 3G, 3I pool ligated plates matching well to well. For pool 3B, 3D, 3F, 3H, 3J pool ligated plates with even plates flipped upside down (H12 in A1). Mix together 20 ul from each well of each plate for pooled plate using the Benchsmart. After pooling all samples, vortex and spin down plates then use 4 ul from the pooled plates for PCR templates. We are doing duplicate PCR.
Pool | Plate1 | Plate2 | Plate3 | Plate4 |
|---|---|---|---|---|
3A | ALF_PLT5_2017_GBS | ALF_PLT6_2017_GBS | ALF_PLT7_2017_GBS | ALF_PLT8_2017_GBS |
Pool | Plate1 | Plate2 | Plate3 | Plate4 |
|---|---|---|---|---|
3B | ALF_PLT5_2017_GBS | ALF_PLT6_2017_GBS (FLIPPED) | ALF_PLT7_2017_GBS | ALF_PLT8_2017_GBS (FLIPPED) |
Pool | Plate1 | Plate2 | Plate3 | Plate4 |
|---|---|---|---|---|
3C | ALF_PLT9_2017_GBS | ALF_PLT1_2020_GBS | ALF_PLT2_2020_GBS | ALF_PLT3_2020_GBS |
Pool | Plate1 | Plate2 | Plate3 | Plate4 |
|---|---|---|---|---|
3D | ALF_PLT9_2017_GBS | ALF_PLT1_2020_GBS (FLIPPED) | ALF_PLT2_2020_GBS | ALF_PLT3_2020_GBS (FLIPPED) |
Pool | Plate1 | Plate2 | Plate3 | Plate4 |
|---|---|---|---|---|
3E | ALF_PLT4_2020_GBS | ALF_PLT5_2020_GBS | ALF_PLT6_2020_GBS | ALF_PLT1_2020_END |
Pool | Plate1 | Plate2 | Plate3 | Plate4 |
|---|---|---|---|---|
3F | ALF_PLT4_2020_GBS | ALF_PLT5_2020_GBS (FLIPPED) | ALF_PLT6_2020_GBS | ALF_PLT1_2020_END (FLIPPED) |
Pool | Plate1 | Plate2 | Plate3 | Plate4 |
|---|---|---|---|---|
3G | ALF_PLT2_2020_END | ALF_PLT3_2020_END | ALF_PLT4_2020_END | ALF_PLT5_2020_END |
Pool | Plate1 | Plate2 | Plate3 | Plate4 |
|---|---|---|---|---|
3H | ALF_PLT2_2020_END | ALF_PLT3_2020_END (FLIPPED) | ALF_PLT4_2020_END | ALF_PLT5_2020_END (FLIPPED) |
Pool | Plate1 | Plate2 | Plate3 |
|---|---|---|---|
3I | ALF_PLT6_2020_END | ALF_PLT7_2020_END |