Page Comparison

...

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)

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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.

...

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.

...

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
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:

...

	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

Results:

Average quantities observed for all products:

...

View file

name	GBS_Run3_3A-3D 2.csv

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

Results:

Average quantities observed for all products:

...

View file

name	GBS_Run3_3E-3H.csv

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	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

Results:

Average quantities observed for all products:

...

View file

name	GBS_Run3_3I-3J.csv

Bioanalyzer

BioAnalyzer Chip Set up:

Well 1: GBS_Run3_3I Pool

...

Well 11: GBS_Run3_3J C6

Well 12: GBS_Run3_3J A9

Gel:

...

EPG 3I-3J:

...

EPG 3I-3J Pools:

...

EPG 3I Wells:

...

EPG 3J Wells:

...

Versions Compared

Old Version 12

New Version Current

Key

Normalize templates

Restriction Digestion_GBS_Run3_Part1

Adaptor Ligation_GBS_Run3_Part1

Restriction Digestion_GBS_Run3_Part2

Adaptor Ligation_GBS_Run3_Part2

PCR Amplification_GBS_Run3

PCR1_3A-3D:

Extra PCR

Comparisons_GBS_Run3_3A-3D

qPCR

Results:

Bioanalyzer

PCR1_3E-3H:

Extra PCR

Comparisons_GBS_Run3_3E-3H

qPCR

Results:

Bioanalyzer

PCR1_3I-3J:

Extra PCR

Comparisons_GBS_Run3_3I-3J

qPCR

Results:

Bioanalyzer

Page Comparison

Versions Compared

Old Version 12

New Version Current

Key

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Restriction Digestion_GBS_Run3_Part1

Adaptor Ligation_GBS_Run3_Part1

Restriction Digestion_GBS_Run3_Part2

Adaptor Ligation_GBS_Run3_Part2

PCR Amplification_GBS_Run3

PCR1_3A-3D:

Extra PCR

Comparisons_GBS_Run3_3A-3D

qPCR

Results:

Bioanalyzer

PCR1_3E-3H:

Extra PCR

Comparisons_GBS_Run3_3E-3H

qPCR

Results:

Bioanalyzer

PCR1_3I-3J:

Extra PCR

Comparisons_GBS_Run3_3I-3J

qPCR

Results:

Bioanalyzer