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

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

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

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

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

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

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

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

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

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

Add 16 ul of PCR1 MM to each well of four new hard shell PCR plates.

Add 4uL of template from pooled plates.

Seal, Vortex, Spin down, and then run on Thermocycler program: GBS1:

Temp C	Cycles	Time

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

Reagent	ul/rxn	rxns	ul needed
5x Iproof buffer	0.425	460	195.5
10 mM dNTPs	0.4	460	184
Primers	1.33	460	611.8
Total	2.155	460	991.3

Then continue with the last four unlisted steps for GBS1 from above:

Temp C	Cycles	Time

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

	1	2	3	4	5	6	7	8	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	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	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	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	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	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	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

Combine 5uL from each well of GBS_Run3_3A into a pool. Make a 1:1000 dilution of this pool to run on qPCR.
Combine 5uL from each well of GBS_Run3_3B into a pool. Make a 1:1000 dilution of this pool to run on qPCR.
Combine 5uL from each well of GBS_Run3_3C into a pool. Make a 1:1000 dilution of this pool to run on qPCR.
Combine 5uL from each well of GBS_Run3_3D into a pool. Make a 1:1000 dilution of this pool to run on qPCR.
After 1:1000 pools have been made for each PCR plate, combine 3A and 3B undiluted pools together into one tube and label GBS_Run3AB_Pool. Make a 1:1000 dilution of this pool to run on qPCR.
After 1:1000 pools have been made for each PCR plate, combine 3C and 3D undiluted pools together into one tube and label GBS_Run3CD_Pool. Make a 1:1000 dilution of this pool to run on qPCR.
Add 16 ul of Illumina Library Quantification MasterMix to each well:

ul/rxn	Reagent	# of rxns	ul needed

ul/rxn	Reagent	# of rxns	ul needed
10 ul	KAPA SYBR FAST qPCR MM (2X)	110	1100
2 ul	Primer Premix (10X)	110	220
4 ul	Ultra Pure Water	110	440
16 ul	Total Volume	110	1760

Add 4 ul of template, pool, or standards to each well:

	1	2	3	4	5	6	7	8	9	10	11	12

	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:

GBS_Run3_3A_Col1: 216.38 nanomoles

GBS_Run3_3A_Col6: 256.31 nanomoles

GBS_Run3_3B_Col1: 222.92 nanomoles

GBS_Run3_3B_Col6: 223.83 nanomoles

GBS_Run3_3C_Col1: 225.86 nanomoles

GBS_Run3_3C_Col6: 227.98 nanomoles

GBS_Run3_3D_Col1: 210.1 nanomoles

GBS_Run3_3D_Col6: 252.31 nanomoles

GBS_Run3_3A Pool: 258.54 nanomoles

GBS_Run3_3B Pool: 246.36 nanomoles

GBS_Run3_3C Pool: 244.65 nanomoles

GBS_Run3_3D Pool: 202.94 nanomoles

GBS_Run3_3AB Pool: 248.88 nanomoles

GBS_Run3_3CD Pool: 207.51 nanomoles

The full result report can be viewed below:

Bioanalyzer

BioAnalyzer Chip Set up:

Well 1: GBS_Run3_3A Pool

Well 2: GBS_Run3_3B Pool

Well 3: GBS_Run3_3C Pool

Well 4: GBS_Run3_3D Pool

Well 5: GBS_Run3_3AB Pool

Well 6: GBS_Run3_3CD Pool

Well 7: GBS_Run3_3A C1

Well 8: GBS_Run3_3A G9

Well 9: GBS_Run3_3B C1

Well 10: GBS_Run3_3B G9

Well 11: GBS_Run3_3C D5

Well 12: GBS_Run3_3D D5

Gel:

Electropherogram:

EPG 3A, 3B, 3C, 3D Pools:

EPG 3AB-3CD Pools:

EPG 3A & 3B Well Comparison:

EPG 3C & 3D Well Comparison:

PCR1_3E-3H:

Reagent	ul/rxn	rxns	ul needed

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

Add 16 ul of PCR1 MM to each well of four new hard shell PCR plates.

Add 4uL of template from pooled plates.

Seal, Vortex, Spin down, and then run on Thermocycler program: GBS1:

Temp C	Cycles	Time

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

Reagent	ul/rxn	rxns	ul needed
5x Iproof buffer	0.425	460	195.5
10 mM dNTPs	0.4	460	184
Primers	1.33	460	611.8
Total	2.155	460	991.3

Then continue with the last four unlisted steps for GBS1 from above:

Temp C	Cycles	Time

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

	1	2	3	4	5	6	7	8	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	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	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	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	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	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	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

Combine 5uL from each well of GBS_Run3_3E into a pool. Make a 1:1000 dilution of this pool to run on qPCR.
Combine 5uL from each well of GBS_Run3_3F into a pool. Make a 1:1000 dilution of this pool to run on qPCR.
Combine 5uL from each well of GBS_Run3_3G into a pool. Make a 1:1000 dilution of this pool to run on qPCR.
Combine 5uL from each well of GBS_Run3_3H into a pool. Make a 1:1000 dilution of this pool to run on qPCR.
After 1:1000 pools have been made for each PCR plate, combine 3E and 3F undiluted pools together into one tube and label GBS_Run3EF_Pool. Make a 1:1000 dilution of this pool to run on qPCR.
After 1:1000 pools have been made for each PCR plate, combine 3G and 3H undiluted pools together into one tube and label GBS_Run3GH_Pool. Make a 1:1000 dilution of this pool to run on qPCR.
Add 16 ul of Illumina Library Quantification MasterMix to each well:

ul/rxn	Reagent	# of rxns	ul needed

ul/rxn	Reagent	# of rxns	ul needed
10 ul	KAPA SYBR FAST qPCR MM (2X)	110	1100
2 ul	Primer Premix (10X)	110	220
4 ul	Ultra Pure Water	110	440
16 ul	Total Volume	110	1760

Add 4 ul of template, pool, or standards to each well:

	1	2	3	4	5	6	7	8	9	10	11	12

	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:

GBS_Run3_3E_Col1: 117.35 nanomoles

GBS_Run3_3E_Col6: 166.34 nanomoles

GBS_Run3_3F_Col1: 116.57 nanomoles

GBS_Run3_3F_Col6: 121.27 nanomoles

GBS_Run3_3G_Col1: 131.49 nanomoles

GBS_Run3_3G_Col6: 141.38 nanomoles

GBS_Run3_3H_Col1: 128.91 nanomoles

GBS_Run3_3H_Col6: 125.3 nanomoles

GBS_Run3_3E Pool: 155.11 nanomoles

GBS_Run3_3F Pool: 128.89 nanomoles

GBS_Run3_3G Pool: 135.65 nanomoles

GBS_Run3_3H Pool: 137.78 nanomoles

GBS_Run3_3EF Pool: 120.23 nanomoles

GBS_Run3_3GH Pool: 128.13 nanomoles

The full result report can be viewed below:

Bioanalyzer

BioAnalyzer Chip Set up:

Well 1: GBS_Run3_3E Pool

Well 2: GBS_Run3_3F Pool

Well 3: GBS_Run3_3G Pool

Well 4: GBS_Run3_3H Pool

Well 5: GBS_Run3_3EF Pool

Well 6: GBS_Run3_3GH Pool

Well 7: GBS_Run3_3E C1

Well 8: GBS_Run3_3E G9

Well 9: GBS_Run3_3F C1

Well 10: GBS_Run3_3F G9

Well 11: GBS_Run3_3G D5

Well 12: GBS_Run3_3H D5

PCR1_3I-3J:

Reagent	ul/rxn	rxns	ul needed

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 16 ul of PCR1 MM to each well of two new hard shell PCR plates.

Add 4uL of template from pooled plates.

Seal, Vortex, Spin down, and then run on Thermocycler program: GBS1:

Temp C	Cycles	Time

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

Reagent	ul/rxn	rxns	ul needed
5x Iproof buffer	0.425	260	110.5
10 mM dNTPs	0.4	260	104
Primers	1.33	260	345.8
Total	2.155	260	560.3

Then continue with the last four unlisted steps for GBS1 from above:

Temp C	Cycles	Time

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

	1	2	3	4	5	6	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	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	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	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	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	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	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

Combine 5uL from each well of GBS_Run3_3I into a pool. Make a 1:1000 dilution of this pool to run on qPCR.
Combine 5uL from each well of GBS_Run3_3J into a pool. Make a 1:1000 dilution of this pool to run on qPCR.
After 1:1000 pools have been made for each PCR plate, combine 3I and 3J undiluted pools together into one tube and label GBS_Run3IJ_Pool. Make a 1:1000 dilution of this pool to run on qPCR.

Add 16 ul of Illumina Library Quantification MasterMix to each well:

ul/rxn	Reagent	# of rxns	ul needed

ul/rxn	Reagent	# of rxns	ul needed
10 ul	KAPA SYBR FAST qPCR MM (2X)	90	900
2 ul	Primer Premix (10X)	90	180
4 ul	Ultra Pure Water	90	360
16 ul	Total Volume	90	1440

Add 4 ul of template, pool, or standards to each well:

	1	2	3	4	5	6	7	8	9	10	11	12

	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:

GBS_Run3_3I_Col1: 142.1

GBS_Run3_3I_Col6: 173.9

GBS_Run3_3I_Col12: 192.35

GBS_Run3_3J_Col1: 173.05

GBS_Run3_3J_Col6: 236.9

GBS_Run3_3J_Col12: 182.26

GBS_Run3_3I Pool: 241.93

GBS_Run3_3J Pool: 229.64

GBS_Run3_3IJ Pool: 230.58

The full result report can be viewed below:

Bioanalyzer

BioAnalyzer Chip Set up:

Well 1: GBS_Run3_3I Pool

Well 2: GBS_Run3_3J Pool

Well 3: GBS_Run3_3IJ Pool

Well 4: GBS_Run3_3I C1

Well 5: GBS_Run3_3I G4

Well 6: GBS_Run3_3I 7A

Well 7: GBS_Run3_3I 10E

Well 8: GBS_Run3_3I 12D

Well 9: GBS_Run3_3J B2

Well 10: GBS_Run3_3J H5

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: