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Normalize templates
Use plate reader to quantify 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)
...
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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Template | EcoR1 MID plate |
|---|---|
ALF_PLT5_2017_GBS | GBS EcoR1 Plate7 |
ALF_PLT6_2017_GBS | GBS EcoR1 Plate 8 |
ALF_PLT7_2017_GBS | GBS EcoR1 Plate 1 |
ALF_PLT8_2017_GBS | GBS EcoR1 Plate 2 |
ALF_PLT9_2017_GBS | GBS EcoR1 Plate 3 |
ALF_PLT1_2020_GBS | GBS EcoR1 Plate 4 |
ALF_PLT2_2020_GBS | GBS EcoR1 Plate 5 |
ALF_PLT3_2020_GBS | GBS EcoR1 Plate 6 |
ALF_PLT4_2020_GBS | GBS EcoR1 Plate 7 |
ALF_PLT5_2020_GBS | GBS EcoR1 Plate 8 |
Label reaction plates with MID plate used.
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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)
...
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.
...
Template | EcoR1 MID plate |
|---|---|
ALF_PLT6_2020_GBS | GBS EcoR1 Plate 1 |
ALF_PLT1_2020_END | GBS EcoR1 Plate 2 |
ALF_PLT2_2020_END | GBS EcoR1 Plate 3 |
ALF_PLT3_2020_END | GBS EcoR1 Plate 4 |
ALF_PLT4_2020_END | GBS EcoR1 Plate 5 |
ALF_PLT5_2020_END | GBS EcoR1 Plate 6 |
ALF_PLT6_2020_END | GBS EcoR1 Plate 7 |
ALF_PLT7_2020_END | GBS EcoR1 Plate 8 |
ALF_PLT8_2020_END | GBS EcoR1 Plate 1 |
Label reaction plates with MID plate used.
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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) |
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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 |
|---|---|---|
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
...
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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| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
A | GBS_Run3_3A_Col1 | GBS_Run3_3A_Col6 | GBS_Run3_3B_Col1 | GBS_Run3_3B_Col6 | GBS_Run3_3C_Col1 | GBS_Run3_3C_Col6 | GBS_Run3_3D_Col1 | GBS_Run3_3D_Col6 | GBS_Run3_3A Pool | NTC | NTC | NTC |
B | GBS_Run3_3A_Col1 | GBS_Run3_3A_Col6 | GBS_Run3_3B_Col1 | GBS_Run3_3B_Col6 | GBS_Run3_3C_Col1 | GBS_Run3_3C_Col6 | GBS_Run3_3D_Col1 | GBS_Run3_3D_Col6 | GBS_Run3_3B Pool | 0.0002 pM Std | 0.0002 pM Std | 0.0002 pM Std |
C | GBS_Run3_3A_Col1 | GBS_Run3_3A_Col6 | GBS_Run3_3B_Col1 | GBS_Run3_3B_Col6 | GBS_Run3_3C_Col1 | GBS_Run3_3C_Col6 | GBS_Run3_3D_Col1 | GBS_Run3_3D_Col6 | GBS_Run3_3C Pool | 0.002 pM Std | 0.002 pM Std | 0.002 pM Std |
D | GBS_Run3_3A_Col1 | GBS_Run3_3A_Col6 | GBS_Run3_3B_Col1 | GBS_Run3_3B_Col6 | GBS_Run3_3C_Col1 | GBS_Run3_3C_Col6 | GBS_Run3_3D_Col1 | GBS_Run3_3D_Col6 | GBS_Run3_3D Pool | 0.02 pM Std | 0.02 pM Std | 0.02 pM Std |
E | GBS_Run3_3A_Col1 | GBS_Run3_3A_Col6 | GBS_Run3_3B_Col1 | GBS_Run3_3B_Col6 | GBS_Run3_3C_Col1 | GBS_Run3_3C_Col6 | GBS_Run3_3D_Col1 | GBS_Run3_3D_Col6 | GBS_Run3_3AB Pool | 0.2 pM Std | 0.2 pM Std | 0.2 pM Std |
F | GBS_Run3_3A_Col1 | GBS_Run3_3A_Col6 | GBS_Run3_3B_Col1 | GBS_Run3_3B_Col6 | GBS_Run3_3C_Col1 | GBS_Run3_3C_Col6 | GBS_Run3_3D_Col1 | GBS_Run3_3D_Col6 | GBS_Run3_3CD Pool | 2 pM Std | 2 pM Std | 2 pM Std |
G | GBS_Run3_3A_Col1 | GBS_Run3_3A_Col6 | GBS_Run3_3B_Col1 | GBS_Run3_3B_Col6 | GBS_Run3_3C_Col1 | GBS_Run3_3C_Col6 | GBS_Run3_3D_Col1 | GBS_Run3_3D_Col6 | 20 pM Std | 20 pM Std | 20 pM Std | |
H | GBS_Run3_3A_Col1 | GBS_Run3_3A_Col6 | GBS_Run3_3B_Col1 | GBS_Run3_3B_Col6 | GBS_Run3_3C_Col1 | GBS_Run3_3C_Col6 | GBS_Run3_3D_Col1 | GBS_Run3_3D_Col6 |
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Results:
Average quantities observed for all products:
...
| View file | ||
|---|---|---|
|
Bioanalyzer
BioAnalyzer Chip Set up:
Well 1: GBS_Run3_3A Pool
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EPG 3C & 3D Well Comparison:
...
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 |
|---|---|---|
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
...
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:
...
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
A | GBS_Run3_3E_Col1 | GBS_Run3_3E_Col6 | GBS_Run3_3F_Col1 | GBS_Run3_3F_Col6 | GBS_Run3_3G_Col1 | GBS_Run3_3G_Col6 | GBS_Run3_3H_Col1 | GBS_Run3_3H_Col6 | GBS_Run3_3E Pool | NTC | NTC | NTC |
B | GBS_Run3_3E_Col1 | GBS_Run3_3E_Col6 | GBS_Run3_3F_Col1 | GBS_Run3_3F_Col6 | GBS_Run3_3G_Col1 | GBS_Run3_3G_Col6 | GBS_Run3_3H_Col1 | GBS_Run3_3H_Col6 | GBS_Run3_3F Pool | 0.0002 pM Std | 0.0002 pM Std | 0.0002 pM Std |
C | GBS_Run3_3E_Col1 | GBS_Run3_3E_Col6 | GBS_Run3_3F_Col1 | GBS_Run3_3F_Col6 | GBS_Run3_3G_Col1 | GBS_Run3_3G_Col6 | GBS_Run3_3H_Col1 | GBS_Run3_3H_Col6 | GBS_Run3_3G Pool | 0.002 pM Std | 0.002 pM Std | 0.002 pM Std |
D | GBS_Run3_3E_Col1 | GBS_Run3_3E_Col6 | GBS_Run3_3F_Col1 | GBS_Run3_3F_Col6 | GBS_Run3_3G_Col1 | GBS_Run3_3G_Col6 | GBS_Run3_3H_Col1 | GBS_Run3_3H_Col6 | GBS_Run3_3H Pool | 0.02 pM Std | 0.02 pM Std | 0.02 pM Std |
E | GBS_Run3_3E_Col1 | GBS_Run3_3E_Col6 | GBS_Run3_3F_Col1 | GBS_Run3_3F_Col6 | GBS_Run3_3G_Col1 | GBS_Run3_3G_Col6 | GBS_Run3_3H_Col1 | GBS_Run3_3H_Col6 | GBS_Run3_3EF Pool | 0.2 pM Std | 0.2 pM Std | 0.2 pM Std |
F | GBS_Run3_3E_Col1 | GBS_Run3_3E_Col6 | GBS_Run3_3F_Col1 | GBS_Run3_3F_Col6 | GBS_Run3_3G_Col1 | GBS_Run3_3G_Col6 | GBS_Run3_3H_Col1 | GBS_Run3_3H_Col6 | GBS_Run3_3GH Pool | 2 pM Std | 2 pM Std | 2 pM Std |
G | GBS_Run3_3E_Col1 | GBS_Run3_3E_Col6 | GBS_Run3_3F_Col1 | GBS_Run3_3F_Col6 | GBS_Run3_3G_Col1 | GBS_Run3_3G_Col6 | GBS_Run3_3H_Col1 | GBS_Run3_3H_Col6 | 20 pM Std | 20 pM Std | 20 pM Std | |
H | GBS_Run3_3E_Col1 | GBS_Run3_3E_Col6 | GBS_Run3_3F_Col1 | GBS_Run3_3F_Col6 | GBS_Run3_3G_Col1 | GBS_Run3_3G_Col6 | GBS_Run3_3H_Col1 | GBS_Run3_3H_Col6 |
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Results:
Average quantities observed for all products:
...
| View file | ||
|---|---|---|
|
Bioanalyzer
BioAnalyzer Chip Set up:
Well 1: GBS_Run3_3E Pool
...
Well 11: GBS_Run3_3G D5
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 |
|---|---|---|
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
...
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:
...
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
A | GBS_Run3_3I_Col1 | GBS_Run3_3I_Col6 | GBS_Run3_3I_Col12 | GBS_Run3_3J_Col1 | GBS_Run3_3J_Col6 | GBS_Run3_3J_Col12 | GBS_Run3_3I Pool | NTC | NTC | NTC | ||
B | GBS_Run3_3I_Col1 | GBS_Run3_3I_Col6 | GBS_Run3_3I_Col12 | GBS_Run3_3J_Col1 | GBS_Run3_3J_Col6 | GBS_Run3_3J_Col12 | GBS_Run3_3I Pool | 0.0002 pM Std | 0.0002 pM Std | 0.0002 pM Std | ||
C | GBS_Run3_3I_Col1 | GBS_Run3_3I_Col6 | GBS_Run3_3I_Col12 | GBS_Run3_3J_Col1 | GBS_Run3_3J_Col6 | GBS_Run3_3J_Col12 | GBS_Run3_3J Pool | 0.002 pM Std | 0.002 pM Std | 0.002 pM Std | ||
D | GBS_Run3_3I_Col1 | GBS_Run3_3I_Col6 | GBS_Run3_3I_Col12 | GBS_Run3_3J_Col1 | GBS_Run3_3J_Col6 | GBS_Run3_3J_Col12 | GBS_Run3_3J Pool | 0.02 pM Std | 0.02 pM Std | 0.02 pM Std | ||
E | GBS_Run3_3I_Col1 | GBS_Run3_3I_Col6 | GBS_Run3_3I_Col12 | GBS_Run3_3J_Col1 | GBS_Run3_3J_Col6 | GBS_Run3_3J_Col12 | GBS_Run3_3IJ Pool | 0.2 pM Std | 0.2 pM Std | 0.2 pM Std | ||
F | GBS_Run3_3I_Col1 | GBS_Run3_3I_Col6 | GBS_Run3_3I_Col12 | GBS_Run3_3J_Col1 | GBS_Run3_3J_Col6 | GBS_Run3_3J_Col12 | GBS_Run3_3IJ Pool | 2 pM Std | 2 pM Std | 2 pM Std | ||
G | GBS_Run3_3I_Col1 | GBS_Run3_3I_Col6 | GBS_Run3_3I_Col12 | GBS_Run3_3J_Col1 | GBS_Run3_3J_Col6 | GBS_Run3_3J_Col12 | 20 pM Std | 20 pM Std | 20 pM Std | |||
H | GBS_Run3_3I_Col1 | GBS_Run3_3I_Col6 | GBS_Run3_3I_Col12 | GBS_Run3_3J_Col1 | GBS_Run3_3J_Col6 | GBS_Run3_3J_Col12 |
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Results:
Average quantities observed for all products:
...
| View file | ||
|---|---|---|
|
Bioanalyzer
BioAnalyzer Chip Set up:
Well 1: GBS_Run3_3I Pool
...