Normalize templates
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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)
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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.
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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)
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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.
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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 | ALF_PLT8_2020_END |
Pool | Plate1 | Plate2 | Plate3 |
|---|---|---|---|
3J | ALF_PLT6_2020_END | ALF_PLT7_2020_END (FLIPPED) | ALF_PLT8_2020_END |
PCR1_3A-3D:
Reagent | ul/rxn | rxns | ul needed |
|---|---|---|---|
H2O | 9.52 | 450 | 4284 |
5x iProof buffer | 4 | 450 | 1800 |
10 mM dNTPs | 0.4 | 450 | 180 |
50 mM MgCl2 | 0.4 | 450 | 180 |
5 uM Illumina Primers | 1.33 | 450 | 598.5 |
iProof TAQ | 0.2 | 450 | 90 |
DMSO | 0.15 | 450 | 67.5 |
total | 16 | 450 | 7200 |
...
Temp C | Cycles | Time |
|---|---|---|
98 | 1X | 3:00 |
60 | 1X | 2:00 |
72 | 1X | 10:00 |
4 | 1X | 0:00 |
Comparisons_GBS_Run3_3A-3D
qPCR
Make 1:1000 dilutions of column 3 from the PCR plates by adding 1 ul to 999 ul TE in a deep well plate:
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EPG 3C & 3D Well Comparison:
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PCR1_3E-3H:
Reagent | ul/rxn | rxns | ul needed |
|---|---|---|---|
H2O | 9.52 | 450 | 4284 |
5x iProof buffer | 4 | 450 | 1800 |
10 mM dNTPs | 0.4 | 450 | 180 |
50 mM MgCl2 | 0.4 | 450 | 180 |
5 uM Illumina Primers | 1.33 | 450 | 598.5 |
iProof TAQ | 0.2 | 450 | 90 |
DMSO | 0.15 | 450 | 67.5 |
total | 16 | 450 | 7200 |
...
Temp C | Cycles | Time |
|---|---|---|
98 | 1X | 3:00 |
60 | 1X | 2:00 |
72 | 1X | 10:00 |
4 | 1X | 0:00 |
Comparisons_GBS_Run3_3E-3H
qPCR
Make 1:1000 dilutions of column 3 from the PCR plates by adding 1 ul to 999 ul TE in a deep well plate:
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Well 12: GBS_Run3_3H D5
PCR1_3I-3J:
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 |
...
Temp C | Cycles | Time |
|---|---|---|
98 | 1X | 3:00 |
60 | 1X | 2:00 |
72 | 1X | 10:00 |
4 | 1X | 0:00 |
Comparisons_GBS_Run3_3I-3J
qPCR
Make 1:1000 dilutions of column 3 from the PCR plates by adding 1 ul to 999 ul TE in a deep well plate:
...