PCR & qPCR Set-Up/Results - Alfalfa 16S/ITS

*Make Protocol Below for 2017 GBS Plates….

Prepare two ITS and two 16S PCR plates for the following template plates:

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
ALF_PLT1_2020_EPI	ALF_PLT2_2020_EPI	ALF_PLT3_2020_EPI	ALF_PLT4_2020_EPI
ALF_PLT5_2020_EPI	ALF_PLT6_2020_EPI	ALF_PLT7_2020_EPI	ALF_PLT8_2020_EPI
ALF_PLT1_2017_GBS	ALF_PLT2_2017_GBS	ALF_PLT3_2017_GBS	ALF_PLT4_2017_GBS
ALF_PLT5_2017_GBS	ALF_PLT6_2017_GBS	ALF_PLT7_2017_GBS	ALF_PLT8_2017_GBS
ALF_PLT9_2017_GBS	ALF_PLT10_2017_GBS	ALF_PLT11_2017_GBS

MasterMix (make for 16 plates in 50mL conical tube)

ul/rxn	Reagent	# of rxns	ul needed
3	5X Kapa HiFi Buffer	1700	5100
0.45	10M dNTPs	1700	765
0.3	Kapa HiFi HotStart DNA Pol	1700	510
7.25	Ultra Pure H2O	1700	12325
11	Total Volume	1700	18700

Add 11 ul to each well of a hard shell, full skirt plate. Seal with bubble strips and store in refrigerator until needed label “ALF PCR”. (If doing manually, one needs 2 ul of template and 2 ul of the primers).

Plates	16S MIDs	ITS MIDs
ALF_PLT1_2020_END	long16S0A1	longITS0A1
ALF_PLT1_2020_END	long16S0B1	longITS0B1
ALF_PLT2_2020_END	long16S0C1	longITS0C1
ALF_PLT2_2020_END	long16S0D1	longITS0D1
ALF_PLT3_2020_END	long16S0E1	longITS0E1
ALF_PLT3_2020_END	long16S0F1	longITS0F1
ALF_PLT4_2020_END	long16S0G1	longITS0G1
ALF_PLT4_2020_END	long16S0H1	longITS0H1

Run on Thermocycler Program GSAF36:

Temp C	Cycles	Time
95*	1X	3:00*
98	36X	0:30
62	36X	0:30
72	36X	0:30
72	1X	5:00
4	1X	0:00

MagBead Cleanup:

Some plates were cleaned up using the Nimbus platform protocol “AxyPrep MagBead PCR1 No MM”

Manually, it was done:

Equilibrate Beads to room Temperature
Add 24 ul of MagBeads to each well and 15 ul of replicate to same well of replicate
Pipette mix up and down 10 times.
Incubate at RT for 5 minutes
Secure plate on magnet plate; incubate at RT for 5 minutes (until wells are clear)
Remove 65 ul from each well; keep tips to left or right depending on the column to avoid bead pellet.
Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well
Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well
Reaspirate from each well to assure maximum EtOH removal
Allow plate to air dry for 7 minutes.
Remove sample plate from magnet plate.
Add 40 ul TE; pipette mix 10+ times. Incubate 2 minutes at RT.
Place sample plate back on magnet for 5 minutes or until all wells are cleared.
Transfer 40 ul to labeled transparent plate (Plate name_PCR_MIDs)

qPCR ALF_END_RUN1

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	10	11	12
A	ALF_END_PLT1_16S	ALF_END_PLT1_ITS	ALF_END_PLT2_16S	ALF_END_PLT2_ITS	ALF_END_PLT3_16S	ALF_END_PLT3_ITS	ALF_END_PLT4_16S	ALF_END_PLT4_ITS	NTC	NTC	NTC
B	ALF_END_PLT1_16S	ALF_END_PLT1_ITS	ALF_END_PLT2_16S	ALF_END_PLT2_ITS	ALF_END_PLT3_16S	ALF_END_PLT3_ITS	ALF_END_PLT4_16S	ALF_END_PLT4_ITS	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	ALF_END_PLT1_16S	ALF_END_PLT1_ITS	ALF_END_PLT2_16S	ALF_END_PLT2_ITS	ALF_END_PLT3_16S	ALF_END_PLT3_ITS	ALF_END_PLT4_16S	ALF_END_PLT4_ITS	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	ALF_END_PLT1_16S	ALF_END_PLT1_ITS	ALF_END_PLT2_16S	ALF_END_PLT2_ITS	ALF_END_PLT3_16S	ALF_END_PLT3_ITS	ALF_END_PLT4_16S	ALF_END_PLT4_ITS	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	ALF_END_PLT1_16S	ALF_END_PLT1_ITS	ALF_END_PLT2_16S	ALF_END_PLT2_ITS	ALF_END_PLT3_16S	ALF_END_PLT3_ITS	ALF_END_PLT4_16S	ALF_END_PLT4_ITS	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	ALF_END_PLT1_16S	ALF_END_PLT1_ITS	ALF_END_PLT2_16S	ALF_END_PLT2_ITS	ALF_END_PLT3_16S	ALF_END_PLT3_ITS	ALF_END_PLT4_16S	ALF_END_PLT4_ITS	2 pM Std	2 pM Std	2 pM Std
G	ALF_END_PLT1_16S	ALF_END_PLT1_ITS	ALF_END_PLT2_16S	ALF_END_PLT2_ITS	ALF_END_PLT3_16S	ALF_END_PLT3_ITS	ALF_END_PLT4_16S	ALF_END_PLT4_ITS	20 pM Std	20 pM Std	20 pM Std
H	ALF_END_PLT1_16S	ALF_END_PLT1_ITS	ALF_END_PLT2_16S	ALF_END_PLT2_ITS	ALF_END_PLT3_16S	ALF_END_PLT3_ITS	ALF_END_PLT4_16S	ALF_END_PLT4_ITS

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

ul/rxn	Reagent	# of rxns	ul needed
10 ul	KAPA SYBR FAST qPCR MM (2X)	100	1000
2 ul	Primer Premix (10X)	100	200
4 ul	Ultra Pure Water	100	400
16 ul	Total Volume	100	1600

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

	1	2	3	4	5	6	7	8	10	11	12
A	ALF_END_PLT1_16S	ALF_END_PLT1_ITS	ALF_END_PLT2_16S	ALF_END_PLT2_ITS	ALF_END_PLT3_16S	ALF_END_PLT3_ITS	ALF_END_PLT4_16S	ALF_END_PLT4_ITS	NTC	NTC	NTC
B	ALF_END_PLT1_16S	ALF_END_PLT1_ITS	ALF_END_PLT2_16S	ALF_END_PLT2_ITS	ALF_END_PLT3_16S	ALF_END_PLT3_ITS	ALF_END_PLT4_16S	ALF_END_PLT4_ITS	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	ALF_END_PLT1_16S	ALF_END_PLT1_ITS	ALF_END_PLT2_16S	ALF_END_PLT2_ITS	ALF_END_PLT3_16S	ALF_END_PLT3_ITS	ALF_END_PLT4_16S	ALF_END_PLT4_ITS	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	ALF_END_PLT1_16S	ALF_END_PLT1_ITS	ALF_END_PLT2_16S	ALF_END_PLT2_ITS	ALF_END_PLT3_16S	ALF_END_PLT3_ITS	ALF_END_PLT4_16S	ALF_END_PLT4_ITS	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	ALF_END_PLT1_16S	ALF_END_PLT1_ITS	ALF_END_PLT2_16S	ALF_END_PLT2_ITS	ALF_END_PLT3_16S	ALF_END_PLT3_ITS	ALF_END_PLT4_16S	ALF_END_PLT4_ITS	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	ALF_END_PLT1_16S	ALF_END_PLT1_ITS	ALF_END_PLT2_16S	ALF_END_PLT2_ITS	ALF_END_PLT3_16S	ALF_END_PLT3_ITS	ALF_END_PLT4_16S	ALF_END_PLT4_ITS	2 pM Std	2 pM Std	2 pM Std
G	ALF_END_PLT1_16S	ALF_END_PLT1_ITS	ALF_END_PLT2_16S	ALF_END_PLT2_ITS	ALF_END_PLT3_16S	ALF_END_PLT3_ITS	ALF_END_PLT4_16S	ALF_END_PLT4_ITS	20 pM Std	20 pM Std	20 pM Std
H	ALF_END_PLT1_16S	ALF_END_PLT1_ITS	ALF_END_PLT2_16S	ALF_END_PLT2_ITS	ALF_END_PLT3_16S	ALF_END_PLT3_ITS	ALF_END_PLT4_16S	ALF_END_PLT4_ITS

Results:

Average results for the following plates (column 3):

ALF_END_PLT1_16S:

ALF_END_PLT1_ITS:

ALF_END_PLT2_16S:

ALF_END_PLT2_ITS:

ALF_END_PLT3_16S:

ALF_END_PLT3_ITS:

ALF_END_PLT4_16S:

ALF_END_PLT4_ITS:

The full result report can be viewed below:

MasterMix (make for 16 plates in 50mL conical tube)

ul/rxn	Reagent	# of rxns	ul needed
3	5X Kapa HiFi Buffer	1700	5100
0.45	10M dNTPs	1700	765
0.3	Kapa HiFi HotStart DNA Pol	1700	510
7.25	Ultra Pure H2O	1700	12325
11	Total Volume	1700	18700

Add 11 ul to each well of a hard shell, full skirt plate. Seal with bubble strips and store in refrigerator until needed label “ALF PCR”. (If doing manually, one needs 2 ul of template and 2 ul of the primers).

Plates	16S MIDs	ITS MIDs
ALF_PLT5_2020_END	long16S0A2	longITS0A2
ALF_PLT5_2020_END	long16S0B2	longITS0B2
ALF_PLT6_2020_END	long16S0C2	longITS0C2
ALF_PLT6_2020_END	long16S0D2	longITS0D2
ALF_PLT7_2020_END	long16S0E2	longITS0E2
ALF_PLT7_2020_END	long16S0F2	longITS0F2
ALF_PLT8_2020_END	long16S0G2	longITS0G2
ALF_PLT8_2020_END	long16S0H2	longITS0H2

Run on Thermocycler Program GSAF36:

Temp C	Cycles	Time
95*	1X	3:00*
98	36X	0:30
62	36X	0:30
72	36X	0:30
72	1X	5:00
4	1X	0:00

MagBead Cleanup:

Some plates were cleaned up using the Nimbus platform protocol “AxyPrep MagBead PCR1 No MM”

Manually, it was done:

Equilibrate Beads to room Temperature
Add 24 ul of MagBeads to each well and 15 ul of replicate to same well of replicate
Pipette mix up and down 10 times.
Incubate at RT for 5 minutes
Secure plate on magnet plate; incubate at RT for 5 minutes (until wells are clear)
Remove 65 ul from each well; keep tips to left or right depending on the column to avoid bead pellet.
Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well
Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well
Reaspirate from each well to assure maximum EtOH removal
Allow plate to air dry for 7 minutes.
Remove sample plate from magnet plate.
Add 40 ul TE; pipette mix 10+ times. Incubate 2 minutes at RT.
Place sample plate back on magnet for 5 minutes or until all wells are cleared.
Transfer 40 ul to labeled transparent plate (Plate name_PCR_MIDs)

qPCR ALF_END_RUN2

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	11	12
A	ALF_END_PLT5_16S	ALF_END_PLT5_ITS	ALF_END_PLT6_16S	ALF_END_PLT6_ITS	ALF_END_PLT7_16S	ALF_END_PLT7_ITS	ALF_END_PLT8_16S	ALF_END_PLT8_ITS		NTC
B	ALF_END_PLT5_16S	ALF_END_PLT5_ITS	ALF_END_PLT6_16S	ALF_END_PLT6_ITS	ALF_END_PLT7_16S	ALF_END_PLT7_ITS	ALF_END_PLT8_16S	ALF_END_PLT8_ITS	04.0002 pM Std	0.0002 pM Std
C	ALF_END_PLT5_16S	ALF_END_PLT5_ITS	ALF_END_PLT6_16S	ALF_END_PLT6_ITS	ALF_END_PLT7_16S	ALF_END_PLT7_ITS	ALF_END_PLT8_16S	ALF_END_PLT8_ITS	0.002 pM Std	0.002 pM Std
D	ALF_END_PLT5_16S	ALF_END_PLT5_ITS	ALF_END_PLT6_16S	ALF_END_PLT6_ITS	ALF_END_PLT7_16S	ALF_END_PLT7_ITS	ALF_END_PLT8_16S	ALF_END_PLT8_ITS	0.02 pM Std	0.02 pM Std
E	ALF_END_PLT5_16S	ALF_END_PLT5_ITS	ALF_END_PLT6_16S	ALF_END_PLT6_ITS	ALF_END_PLT7_16S	ALF_END_PLT7_ITS	ALF_END_PLT8_16S	ALF_END_PLT8_ITS	0.2 pM Std	0.2 pM Std
F	ALF_END_PLT5_16S	ALF_END_PLT5_ITS	ALF_END_PLT6_16S	ALF_END_PLT6_ITS	ALF_END_PLT7_16S	ALF_END_PLT7_ITS	ALF_END_PLT8_16S	ALF_END_PLT8_ITS	2 pM Std	2 pM Std
G	ALF_END_PLT5_16S	ALF_END_PLT5_ITS	ALF_END_PLT6_16S	ALF_END_PLT6_ITS	ALF_END_PLT7_16S	ALF_END_PLT7_ITS	ALF_END_PLT8_16S	ALF_END_PLT8_ITS	20 pM Std	20 pM Std
H	ALF_END_PLT5_16S	ALF_END_PLT5_ITS	ALF_END_PLT6_16S	ALF_END_PLT6_ITS	ALF_END_PLT7_16S	ALF_END_PLT7_ITS	ALF_END_PLT8_16S	ALF_END_PLT8_ITS

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

ul/rxn	Reagent	# of rxns	ul needed
10 ul	KAPA SYBR FAST qPCR MM (2X)	100	1000
2 ul	Primer Premix (10X)	100	200
4 ul	Ultra Pure Water	100	400
16 ul	Total Volume	100	1600

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

	1	2	3	4	5	6	7	8	10	11	12
A	ALF_END_PLT5_16S	ALF_END_PLT5_ITS	ALF_END_PLT6_16S	ALF_END_PLT6_ITS	ALF_END_PLT7_16S	ALF_END_PLT7_ITS	ALF_END_PLT8_16S	ALF_END_PLT8_ITS	NTC	NTC	NTC
B	ALF_END_PLT5_16S	ALF_END_PLT5_ITS	ALF_END_PLT6_16S	ALF_END_PLT6_ITS	ALF_END_PLT7_16S	ALF_END_PLT7_ITS	ALF_END_PLT8_16S	ALF_END_PLT8_ITS	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	ALF_END_PLT5_16S	ALF_END_PLT5_ITS	ALF_END_PLT6_16S	ALF_END_PLT6_ITS	ALF_END_PLT7_16S	ALF_END_PLT7_ITS	ALF_END_PLT8_16S	ALF_END_PLT8_ITS	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	ALF_END_PLT5_16S	ALF_END_PLT5_ITS	ALF_END_PLT6_16S	ALF_END_PLT6_ITS	ALF_END_PLT7_16S	ALF_END_PLT7_ITS	ALF_END_PLT8_16S	ALF_END_PLT8_ITS	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	ALF_END_PLT5_16S	ALF_END_PLT5_ITS	ALF_END_PLT6_16S	ALF_END_PLT6_ITS	ALF_END_PLT7_16S	ALF_END_PLT7_ITS	ALF_END_PLT8_16S	ALF_END_PLT8_ITS	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	ALF_END_PLT5_16S	ALF_END_PLT5_ITS	ALF_END_PLT6_16S	ALF_END_PLT6_ITS	ALF_END_PLT7_16S	ALF_END_PLT7_ITS	ALF_END_PLT8_16S	ALF_END_PLT8_ITS	2 pM Std	2 pM Std	2 pM Std
G	ALF_END_PLT5_16S	ALF_END_PLT5_ITS	ALF_END_PLT6_16S	ALF_END_PLT6_ITS	ALF_END_PLT7_16S	ALF_END_PLT7_ITS	ALF_END_PLT8_16S	ALF_END_PLT8_ITS	20 pM Std	20 pM Std	20 pM Std
H	ALF_END_PLT5_16S	ALF_END_PLT5_ITS	ALF_END_PLT6_16S	ALF_END_PLT6_ITS	ALF_END_PLT7_16S	ALF_END_PLT7_ITS	ALF_END_PLT8_16S	ALF_END_PLT8_ITS

Results:

Average results for the following plates (column 3):

ALF_END_PLT5_16S:

ALF_END_PLT5_ITS:

ALF_END_PLT6_16S:

ALF_END_PLT6_ITS:

ALF_END_PLT7_16S:

ALF_END_PLT7_ITS:

ALF_END_PLT8_16S:

ALF_END_PLT8_ITS:

The full result report can be viewed below:

MasterMix (make for 16 plates in 50mL conical tube)

ul/rxn	Reagent	# of rxns	ul needed
3	5X Kapa HiFi Buffer	1700	5100
0.45	10M dNTPs	1700	765
0.3	Kapa HiFi HotStart DNA Pol	1700	510
7.25	Ultra Pure H2O	1700	12325
11	Total Volume	1700	18700

Add 11 ul to each well of a hard shell, full skirt plate. Seal with bubble strips and store in refrigerator until needed label “ALF PCR”. (If doing manually, one needs 2 ul of template and 2 ul of the primers).

Plates	16S MIDs	ITS MIDs
ALF_PLT1_2020_EPI	long16S0A3	longITS0A3
ALF_PLT1_2020_EPI	long16S0B3	longITS0B3
ALF_PLT2_2020_EPI	long16S0C3	longITS0C3
ALF_PLT2_2020_EPI	long16S0D3	longITS0D3
ALF_PLT3_2020_EPI	long16S0E3	longITS0E3
ALF_PLT3_2020_EPI	long16S0F3	longITS0F3
ALF_PLT4_2020_EPI	long16S0G3	longITS0G3
ALF_PLT4_2020_EPI	long16S0H3	longITS0H3

Run on Thermocycler Program GSAF36:

Temp C	Cycles	Time
95*	1X	3:00*
98	36X	0:30
62	36X	0:30
72	36X	0:30
72	1X	5:00
4	1X	0:00

MagBead Cleanup:

Some plates were cleaned up using the Nimbus platform protocol “AxyPrep MagBead PCR1 No MM”

Manually, it was done:

Equilibrate Beads to room Temperature
Add 24 ul of MagBeads to each well and 15 ul of replicate to same well of replicate
Pipette mix up and down 10 times.
Incubate at RT for 5 minutes
Secure plate on magnet plate; incubate at RT for 5 minutes (until wells are clear)
Remove 65 ul from each well; keep tips to left or right depending on the column to avoid bead pellet.
Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well
Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well
Reaspirate from each well to assure maximum EtOH removal
Allow plate to air dry for 7 minutes.
Remove sample plate from magnet plate.
Add 40 ul TE; pipette mix 10+ times. Incubate 2 minutes at RT.
Place sample plate back on magnet for 5 minutes or until all wells are cleared.
Transfer 40 ul to labeled transparent plate (Plate name_PCR_MIDs)

qPCR ALF_EPI_RUN1

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	11	12
A	ALF_EPI_PLT1_16S	ALF_EPI_PLT1_ITS	ALF_EPI_PLT2_16S	ALF_EPI_PLT2_ITS	ALF_EPI_PLT3_16S	ALF_EPI_PLT3_ITS	ALF_EPI_PLT4_16S	ALF_EPI_PLT4_ITS		NTC
B	ALF_EPI_PLT1_16S	ALF_EPI_PLT1_ITS	ALF_EPI_PLT2_16S	ALF_EPI_PLT2_ITS	ALF_EPI_PLT3_16S	ALF_EPI_PLT3_ITS	ALF_EPI_PLT4_16S	ALF_EPI_PLT4_ITS	04.0002 pM Std	0.0002 pM Std
C	ALF_EPI_PLT1_16S	ALF_EPI_PLT1_ITS	ALF_EPI_PLT2_16S	ALF_EPI_PLT2_ITS	ALF_EPI_PLT3_16S	ALF_EPI_PLT3_ITS	ALF_EPI_PLT4_16S	ALF_EPI_PLT4_ITS	0.002 pM Std	0.002 pM Std
D	ALF_EPI_PLT1_16S	ALF_EPI_PLT1_ITS	ALF_EPI_PLT2_16S	ALF_EPI_PLT2_ITS	ALF_EPI_PLT3_16S	ALF_EPI_PLT3_ITS	ALF_EPI_PLT4_16S	ALF_EPI_PLT4_ITS	0.02 pM Std	0.02 pM Std
E	ALF_EPI_PLT1_16S	ALF_EPI_PLT1_ITS	ALF_EPI_PLT2_16S	ALF_EPI_PLT2_ITS	ALF_EPI_PLT3_16S	ALF_EPI_PLT3_ITS	ALF_EPI_PLT4_16S	ALF_EPI_PLT4_ITS	0.2 pM Std	0.2 pM Std
F	ALF_EPI_PLT1_16S	ALF_EPI_PLT1_ITS	ALF_EPI_PLT2_16S	ALF_EPI_PLT2_ITS	ALF_EPI_PLT3_16S	ALF_EPI_PLT3_ITS	ALF_EPI_PLT4_16S	ALF_EPI_PLT4_ITS	2 pM Std	2 pM Std
G	ALF_EPI_PLT1_16S	ALF_EPI_PLT1_ITS	ALF_EPI_PLT2_16S	ALF_EPI_PLT2_ITS	ALF_EPI_PLT3_16S	ALF_EPI_PLT3_ITS	ALF_EPI_PLT4_16S	ALF_EPI_PLT4_ITS	20 pM Std	20 pM Std
H	ALF_EPI_PLT1_16S	ALF_EPI_PLT1_ITS	ALF_EPI_PLT2_16S	ALF_EPI_PLT2_ITS	ALF_EPI_PLT3_16S	ALF_EPI_PLT3_ITS	ALF_EPI_PLT4_16S	ALF_EPI_PLT4_ITS

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

ul/rxn	Reagent	# of rxns	ul needed
10 ul	KAPA SYBR FAST qPCR MM (2X)	100	1000
2 ul	Primer Premix (10X)	100	200
4 ul	Ultra Pure Water	100	400
16 ul	Total Volume	100	1600

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

	1	2	3	4	5	6	7	8	10	11	12
A	ALF_EPI_PLT1_16S	ALF_EPI_PLT1_ITS	ALF_EPI_PLT2_16S	ALF_EPI_PLT2_ITS	ALF_EPI_PLT3_16S	ALF_EPI_PLT3_ITS	ALF_EPI_PLT4_16S	ALF_EPI_PLT4_ITS	NTC	NTC	NTC
B	ALF_EPI_PLT1_16S	ALF_EPI_PLT1_ITS	ALF_EPI_PLT2_16S	ALF_EPI_PLT2_ITS	ALF_EPI_PLT3_16S	ALF_EPI_PLT3_ITS	ALF_EPI_PLT4_16S	ALF_EPI_PLT4_ITS	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	ALF_EPI_PLT1_16S	ALF_EPI_PLT1_ITS	ALF_EPI_PLT2_16S	ALF_EPI_PLT2_ITS	ALF_EPI_PLT3_16S	ALF_EPI_PLT3_ITS	ALF_EPI_PLT4_16S	ALF_EPI_PLT4_ITS	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	ALF_EPI_PLT1_16S	ALF_EPI_PLT1_ITS	ALF_EPI_PLT2_16S	ALF_EPI_PLT2_ITS	ALF_EPI_PLT3_16S	ALF_EPI_PLT3_ITS	ALF_EPI_PLT4_16S	ALF_EPI_PLT4_ITS	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	ALF_EPI_PLT1_16S	ALF_EPI_PLT1_ITS	ALF_EPI_PLT2_16S	ALF_EPI_PLT2_ITS	ALF_EPI_PLT3_16S	ALF_EPI_PLT3_ITS	ALF_EPI_PLT4_16S	ALF_EPI_PLT4_ITS	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	ALF_EPI_PLT1_16S	ALF_EPI_PLT1_ITS	ALF_EPI_PLT2_16S	ALF_EPI_PLT2_ITS	ALF_EPI_PLT3_16S	ALF_EPI_PLT3_ITS	ALF_EPI_PLT4_16S	ALF_EPI_PLT4_ITS	2 pM Std	2 pM Std	2 pM Std
G	ALF_EPI_PLT1_16S	ALF_EPI_PLT1_ITS	ALF_EPI_PLT2_16S	ALF_EPI_PLT2_ITS	ALF_EPI_PLT3_16S	ALF_EPI_PLT3_ITS	ALF_EPI_PLT4_16S	ALF_EPI_PLT4_ITS	20 pM Std	20 pM Std	20 pM Std
H	ALF_EPI_PLT1_16S	ALF_EPI_PLT1_ITS	ALF_EPI_PLT2_16S	ALF_EPI_PLT2_ITS	ALF_EPI_PLT3_16S	ALF_EPI_PLT3_ITS	ALF_EPI_PLT4_16S	ALF_EPI_PLT4_ITS

Results:

Average results for the following plates (column 3):

ALF_EPI_PLT1_16S:

ALF_EPI_PLT1_ITS:

ALF_EPI_PLT2_16S:

ALF_EPI_PLT2_ITS:

ALF_EPI_PLT3_16S:

ALF_EPI_PLT3_ITS:

ALF_EPI_PLT4_16S:

ALF_EPI_PLT4_ITS:

The full result report can be viewed below:

MasterMix (make for 16 plates in 50mL conical tube)

ul/rxn	Reagent	# of rxns	ul needed
3	5X Kapa HiFi Buffer	1700	5100
0.45	10M dNTPs	1700	765
0.3	Kapa HiFi HotStart DNA Pol	1700	510
7.25	Ultra Pure H2O	1700	12325
11	Total Volume	1700	18700

Add 11 ul to each well of a hard shell, full skirt plate. Seal with bubble strips and store in refrigerator until needed label “ALF PCR”. (If doing manually, one needs 2 ul of template and 2 ul of the primers).

Plates	16S MIDs	ITS MIDs
ALF_PLT5_2020_EPI	long16S0A4	longITS0A4
ALF_PLT5_2020_EPI	long16S0B4	longITS0B4
ALF_PLT6_2020_EPI	long16S0C4	longITS0C4
ALF_PLT6_2020_EPI	long16S0D4	longITS0D4
ALF_PLT7_2020_EPI	long16S0E4	longITS0E4
ALF_PLT7_2020_EPI	long16S0F4	longITS0F4
ALF_PLT8_2020_EPI	long16S0G4	longITS0G4
ALF_PLT8_2020_EPI	long16S0H4	longITS0H4

Run on Thermocycler Program GSAF36:

Temp C	Cycles	Time
95*	1X	3:00*
98	36X	0:30
62	36X	0:30
72	36X	0:30
72	1X	5:00
4	1X	0:00

MagBead Cleanup:

Some plates were cleaned up using the Nimbus platform protocol “AxyPrep MagBead PCR1 No MM”

Manually, it was done:

Equilibrate Beads to room Temperature
Add 24 ul of MagBeads to each well and 15 ul of replicate to same well of replicate
Pipette mix up and down 10 times.
Incubate at RT for 5 minutes
Secure plate on magnet plate; incubate at RT for 5 minutes (until wells are clear)
Remove 65 ul from each well; keep tips to left or right depending on the column to avoid bead pellet.
Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well
Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well
Reaspirate from each well to assure maximum EtOH removal
Allow plate to air dry for 7 minutes.
Remove sample plate from magnet plate.
Add 40 ul TE; pipette mix 10+ times. Incubate 2 minutes at RT.
Place sample plate back on magnet for 5 minutes or until all wells are cleared.
Transfer 40 ul to labeled transparent plate (Plate name_PCR_MIDs)

qPCR ALF_EPI_RUN2

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	10	11	12
A	ALF_EPI_PLT5_16S	ALF_EPI_PLT5_ITS	ALF_EPI_PLT6_16S	ALF_EPI_PLT6_ITS	ALF_EPI_PLT7_16S	ALF_EPI_PLT7_ITS	ALF_EPI_PLT8_16S	ALF_EPI_PLT8_ITS	NTC	NTC	NTC
B	ALF_EPI_PLT5_16S	ALF_EPI_PLT5_ITS	ALF_EPI_PLT6_16S	ALF_EPI_PLT6_ITS	ALF_EPI_PLT7_16S	ALF_EPI_PLT7_ITS	ALF_EPI_PLT8_16S	ALF_EPI_PLT8_ITS	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	ALF_EPI_PLT5_16S	ALF_EPI_PLT5_ITS	ALF_EPI_PLT6_16S	ALF_EPI_PLT6_ITS	ALF_EPI_PLT7_16S	ALF_EPI_PLT7_ITS	ALF_EPI_PLT8_16S	ALF_EPI_PLT8_ITS	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	ALF_EPI_PLT5_16S	ALF_EPI_PLT5_ITS	ALF_EPI_PLT6_16S	ALF_EPI_PLT6_ITS	ALF_EPI_PLT7_16S	ALF_EPI_PLT7_ITS	ALF_EPI_PLT8_16S	ALF_EPI_PLT8_ITS	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	ALF_EPI_PLT5_16S	ALF_EPI_PLT5_ITS	ALF_EPI_PLT6_16S	ALF_EPI_PLT6_ITS	ALF_EPI_PLT7_16S	ALF_EPI_PLT7_ITS	ALF_EPI_PLT8_16S	ALF_EPI_PLT8_ITS	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	ALF_EPI_PLT5_16S	ALF_EPI_PLT5_ITS	ALF_EPI_PLT6_16S	ALF_EPI_PLT6_ITS	ALF_EPI_PLT7_16S	ALF_EPI_PLT7_ITS	ALF_EPI_PLT8_16S	ALF_EPI_PLT8_ITS	2 pM Std	2 pM Std	2 pM Std
G	ALF_EPI_PLT5_16S	ALF_EPI_PLT5_ITS	ALF_EPI_PLT6_16S	ALF_EPI_PLT6_ITS	ALF_EPI_PLT7_16S	ALF_EPI_PLT7_ITS	ALF_EPI_PLT8_16S	ALF_EPI_PLT8_ITS	20 pM Std	20 pM Std	20 pM Std
H	ALF_EPI_PLT5_16S	ALF_EPI_PLT5_ITS	ALF_EPI_PLT6_16S	ALF_EPI_PLT6_ITS	ALF_EPI_PLT7_16S	ALF_EPI_PLT7_ITS	ALF_EPI_PLT8_16S	ALF_EPI_PLT8_ITS

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

ul/rxn	Reagent	# of rxns	ul needed
10 ul	KAPA SYBR FAST qPCR MM (2X)	100	1000
2 ul	Primer Premix (10X)	100	200
4 ul	Ultra Pure Water	100	400
16 ul	Total Volume	100	1600

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

	1	2	3	4	5	6	7	8	10	11	12
A	ALF_EPI_PLT5_16S	ALF_EPI_PLT5_ITS	ALF_EPI_PLT6_16S	ALF_EPI_PLT6_ITS	ALF_EPI_PLT7_16S	ALF_EPI_PLT7_ITS	ALF_EPI_PLT8_16S	ALF_EPI_PLT8_ITS	NTC	NTC	NTC
B	ALF_EPI_PLT5_16S	ALF_EPI_PLT5_ITS	ALF_EPI_PLT6_16S	ALF_EPI_PLT6_ITS	ALF_EPI_PLT7_16S	ALF_EPI_PLT7_ITS	ALF_EPI_PLT8_16S	ALF_EPI_PLT8_ITS	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	ALF_EPI_PLT5_16S	ALF_EPI_PLT5_ITS	ALF_EPI_PLT6_16S	ALF_EPI_PLT6_ITS	ALF_EPI_PLT7_16S	ALF_EPI_PLT7_ITS	ALF_EPI_PLT8_16S	ALF_EPI_PLT8_ITS	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	ALF_EPI_PLT5_16S	ALF_EPI_PLT5_ITS	ALF_EPI_PLT6_16S	ALF_EPI_PLT6_ITS	ALF_EPI_PLT7_16S	ALF_EPI_PLT7_ITS	ALF_EPI_PLT8_16S	ALF_EPI_PLT8_ITS	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	ALF_EPI_PLT5_16S	ALF_EPI_PLT5_ITS	ALF_EPI_PLT6_16S	ALF_EPI_PLT6_ITS	ALF_EPI_PLT7_16S	ALF_EPI_PLT7_ITS	ALF_EPI_PLT8_16S	ALF_EPI_PLT8_ITS	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	ALF_EPI_PLT5_16S	ALF_EPI_PLT5_ITS	ALF_EPI_PLT6_16S	ALF_EPI_PLT6_ITS	ALF_EPI_PLT7_16S	ALF_EPI_PLT7_ITS	ALF_EPI_PLT8_16S	ALF_EPI_PLT8_ITS	2 pM Std	2 pM Std	2 pM Std
G	ALF_EPI_PLT5_16S	ALF_EPI_PLT5_ITS	ALF_EPI_PLT6_16S	ALF_EPI_PLT6_ITS	ALF_EPI_PLT7_16S	ALF_EPI_PLT7_ITS	ALF_EPI_PLT8_16S	ALF_EPI_PLT8_ITS	20 pM Std	20 pM Std	20 pM Std
H	ALF_EPI_PLT5_16S	ALF_EPI_PLT5_ITS	ALF_EPI_PLT6_16S	ALF_EPI_PLT6_ITS	ALF_EPI_PLT7_16S	ALF_EPI_PLT7_ITS	ALF_EPI_PLT8_16S	ALF_EPI_PLT8_ITS

Results:

Average results for the following plates (column 3):

ALF_EPI_PLT5_16S:

ALF_EPI_PLT5_ITS:

ALF_EPI_PLT6_16S:

ALF_EPI_PLT6_ITS:

ALF_EPI_PLT7_16S:

ALF_EPI_PLT7_ITS:

ALF_EPI_PLT8_16S:

ALF_EPI_PLT8_ITS:

The full result report can be viewed below: