NovaSeq6 1-Step PCR and qPCR

Plates in red have been extracted, PCRed, cleaned, quantified, and normalized by 7-15-22. Bolded red have been qPCRed.

NS6 Plates

6AC1	6AC2	6AC3
6BM1	6BM2
6LVD1	6LVD2	6LVD3	6LVD4
6RD1	6RD2	6RD3
6GTL1	6GTL2
6RD4	6RD5	6RD6	6RD7
6LVD5	6LVD6	6LVD7
MP002	MP003	MP006

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

ul/rxn	Reagent	# of rxns	ul needed

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	HPLC 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 “NS6 PCR”. (If doing manually, one needs 2 ul of template and 2 ul of the primers).

Plate	16S Primer	ITS Primer

Plate	16S Primer	ITS Primer
6AC1	16S0A1	ITS0A1
6AC1	16S0B1	ITS0B1
6AC2	16S0C1	ITS0C1
6AC2	16S0D1	ITS0D1
6AC3	16S0E1	ITS0E1
6AC3	16S0F1	ITS0F1
6BM1	16S0G1	ITS0G1
6BM1	16S0H1	ITS0H1

Run on Thermocycler Program GSAF36:

Temp C	Cycles	Time

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)
Store in -20 until ready to qPCR

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

ul/rxn	Reagent	# of rxns	ul needed

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	HPLC 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 labeled “NS5 PCR”. (If doing manually, one needs 2 ul of template and 2 ul of the primers).

Plate	16S Primer	ITS Primer

Plate	16S Primer	ITS Primer
6BM2	16S0A2	ITS0A2
6BM2
6LVD1	16S0C2	ITS0C2
6LVD1	16S0D2	ITS0D2
6LVD2	16S0E2	ITS0E2
6LVD2	16S0F2	ITS0F2
6LVD3	16S0G2	ITS0G2
6LVD3	16S0H2	ITS0H2

Run on Thermocycler Program GSAF36:

Temp C	Cycles	Time

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)
Store in -20 until ready to qPCR.

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

ul/rxn	Reagent	# of rxns	ul needed

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	HPLC 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 labeled “NS5 PCR”. (If doing manually, one needs 2 ul of template and 2 ul of the primers).

Plate	16S Primer	ITS Primer

Plate	16S Primer	ITS Primer
6LVD4	16S0A3	ITS0A3
6LVD4	16S0B3	ITS0B3
6RD1	16S0C3	ITS0C3
6RD1	16S0D3	ITS0D3
6RD2	16S0E3	ITS0E3
6RD2	16S0F3	ITS0F3
6RD3	16S0G3	ITS0G3
6RD3	16S0H3	ITS0H3

Run on Thermocycler Program GSAF36:

Temp C	Cycles	Time

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)
Store in -20 until ready to qPCR.

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

ul/rxn	Reagent	# of rxns	ul needed

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	HPLC 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 labeled “NS5 PCR”. (If doing manually, one needs 2 ul of template and 2 ul of the primers).

Plate	16S Primer	ITS Primer

Plate	16S Primer	ITS Primer
6GTL1	16S0G4	ITS0G4
6GTL1	16S0H4	ITS0H4
6GTL2	16S0A5	ITS0A5
6GTL2	16S0B5	ITS0B5

Run on Thermocycler Program GSAF36:

Temp C	Cycles	Time

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)
Store in -20 until ready to qPCR.

qPCR NovaSeq6_Set1

Make 1:1000 dilutions of column 8 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

	4	5	6	7	8	9	10	11	12
A	2TRNL1_TRNL	2TRNL_16S	6AC1_16S	6AC1_ITS	6AC2_16S	6AC2_ITS	NTC	NTC	NTC
B	2TRNL1_TRNL	2TRNL_16S	6AC1_16S	6AC1_ITS	6AC2_16S	6AC2_ITS	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	2TRNL1_TRNL	2TRNL_16S	6AC1_16S	6AC1_ITS	6AC2_16S	6AC2_ITS	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	2TRNL1_TRNL	2TRNL_16S	6AC1_16S	6AC1_ITS	6AC2_16S	6AC2_ITS	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	2TRNL1_TRNL	2TRNL_16S	6AC1_16S	6AC1_ITS	6AC2_16S	6AC2_ITS	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	2TRNL1_TRNL	2TRNL_16S	6AC1_16S	6AC1_ITS	6AC2_16S	6AC2_ITS	2 pM Std	2 pM Std	2 pM Std
G	2TRNL1_TRNL	2TRNL_16S	6AC1_16S	6AC1_ITS	6AC2_16S	6AC2_ITS	20 pM Std	20 pM Std	20 pM Std
H	2TRNL1_TRNL	2TRNL_16S	6AC1_16S	6AC1_ITS	6AC2_16S	6AC2_ITS

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

	4	5	6	7	8	9	10	11	12
A	2TRNL1_TRNL	2TRNL_16S	6AC1_16S	6AC1_ITS	6AC2_16S	6AC2_ITS	20 pM Std	20 pM Std	20 pM Std
B	2TRNL1_TRNL	2TRNL_16S	6AC1_16S	6AC1_ITS	6AC2_16S	6AC2_ITS	2 pM Std	2 pM Std	2 pM Std
C	2TRNL1_TRNL	2TRNL_16S	6AC1_16S	6AC1_ITS	6AC2_16S	6AC2_ITS	0.2 pM Std	0.2 pM Std	0.2 pM Std
D	2TRNL1_TRNL	2TRNL_16S	6AC1_16S	6AC1_ITS	6AC2_16S	6AC2_ITS	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	2TRNL1_TRNL	2TRNL_16S	6AC1_16S	6AC1_ITS	6AC2_16S	6AC2_ITS	0.002 pM Std	0.002 pM Std	0.002 pM Std
F	2TRNL1_TRNL	2TRNL_16S	6AC1_16S	6AC1_ITS	6AC2_16S	6AC2_ITS	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
G	2TRNL1_TRNL	2TRNL_16S	6AC1_16S	6AC1_ITS	6AC2_16S	6AC2_ITS	NTC	NTC	NTC
H	2TRNL1_TRNL	2TRNL_16S	6AC1_16S	6AC1_ITS	6AC2_16S	6AC2_ITS	NTC	NTC	NTC

Results:

Plate Name	Average Result (nanomoles)

Plate Name	Average Result (nanomoles)
2TRNL1_TRNL	10.63
2TRNL_16S	6.02E-01
6AC1_16S	23.35
6AC1_ITS	36.97
6AC2_16S	67.91
6AC2_ITS	28.37

Full qPCR results below:

qPCR NovaSeq6_Set2

Make 1:1000 dilutions of column 8 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	9	10
A	6AC3_ITS	6AC3_16S	6RD1_ITS	6RD1_16S	6BM1_ITS	6BM1_16S	6LVD1_ITS	6LVD1_16S	6GTL1_ITS	6GTL1_16S
B	6AC3_ITS	6AC3_16S	6RD1_ITS	6RD1_16S	6BM1_ITS	6BM1_16S	6LVD1_ITS	6LVD1_16S	6GTL1_ITS	6GTL1_16S
C	6AC3_ITS	6AC3_16S	6RD1_ITS	6RD1_16S	6BM1_ITS	6BM1_16S	6LVD1_ITS	6LVD1_16S	6GTL1_ITS	6GTL1_16S
D	6AC3_ITS	6AC3_16S	6RD1_ITS	6RD1_16S	6BM1_ITS	6BM1_16S	6LVD1_ITS	6LVD1_16S	6GTL1_ITS	6GTL1_16S
E	6AC3_ITS	6AC3_16S	6RD1_ITS	6RD1_16S	6BM1_ITS	6BM1_16S	6LVD1_ITS	6LVD1_16S	6GTL1_ITS	6GTL1_16S
F	6AC3_ITS	6AC3_16S	6RD1_ITS	6RD1_16S	6BM1_ITS	6BM1_16S	6LVD1_ITS	6LVD1_16S	6GTL1_ITS	6GTL1_16S
G	6AC3_ITS	6AC3_16S	6RD1_ITS	6RD1_16S	6BM1_ITS	6BM1_16S	6LVD1_ITS	6LVD1_16S	6GTL1_ITS	6GTL1_16S
H	6AC3_ITS	6AC3_16S	6RD1_ITS	6RD1_16S	6BM1_ITS	6BM1_16S	6LVD1_ITS	6LVD1_16S	6GTL1_ITS	6GTL1_16S

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	6AC3_ITS	6AC3_16S	6RD1_ITS	6RD1_16S	6BM1_ITS	6BM1_16S	6LVD1_ITS	6LVD1_16S	6GTL1_ITS	6GTL1_16S	20 pM Std	20 pM Std
B	6AC3_ITS	6AC3_16S	6RD1_ITS	6RD1_16S	6BM1_ITS	6BM1_16S	6LVD1_ITS	6LVD1_16S	6GTL1_ITS	6GTL1_16S	2 pM Std	2 pM Std
C	6AC3_ITS	6AC3_16S	6RD1_ITS	6RD1_16S	6BM1_ITS	6BM1_16S	6LVD1_ITS	6LVD1_16S	6GTL1_ITS	6GTL1_16S	0.2 pM Std	0.2 pM Std
D	6AC3_ITS	6AC3_16S	6RD1_ITS	6RD1_16S	6BM1_ITS	6BM1_16S	6LVD1_ITS	6LVD1_16S	6GTL1_ITS	6GTL1_16S	0.02 pM Std	0.02 pM Std
E	6AC3_ITS	6AC3_16S	6RD1_ITS	6RD1_16S	6BM1_ITS	6BM1_16S	6LVD1_ITS	6LVD1_16S	6GTL1_ITS	6GTL1_16S	0.002 pM Std	0.002 pM Std
F	6AC3_ITS	6AC3_16S	6RD1_ITS	6RD1_16S	6BM1_ITS	6BM1_16S	6LVD1_ITS	6LVD1_16S	6GTL1_ITS	6GTL1_16S	0.0002 pM Std	0.0002 pM Std
G	6AC3_ITS	6AC3_16S	6RD1_ITS	6RD1_16S	6BM1_ITS	6BM1_16S	6LVD1_ITS	6LVD1_16S	6GTL1_ITS	6GTL1_16S	NTC	NTC
H	6AC3_ITS	6AC3_16S	6RD1_ITS	6RD1_16S	6BM1_ITS	6BM1_16S	6LVD1_ITS	6LVD1_16S	6GTL1_ITS	6GTL1_16S	NTC	NTC

Results:

Plate Name	Average Result (nanomoles)

Plate Name	Average Result (nanomoles)
6AC3_ITS	4.125
6AC3_16S	7.46
6RD1_ITS	10.152
6RD1_16S	15.709
6BM1_ITS	7.874
6BM1_16S	4.842
6LVD1_ITS	4.532
6LVD1_16S	8.102
6GTL1_ITS	5.992
6GTL1_16S	Needs to be redone

Full qPCR results below:

qPCR NovaSeq6_Set3

Make 1:1000 dilutions of column 8 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	9	10	11	12
A	6LVD2_ITS	6LVD2_16S	2TRNL3_16S	2TRNL3_TRNL	2TRNL2_TRNL	2TRNL2_16S	6RD2_16S	6RD2_ITS	6GTL1_16S	2TRNL1_16S_2		NTC
B	6LVD2_ITS	6LVD2_16S	2TRNL3_16S	2TRNL3_TRNL	2TRNL2_TRNL	2TRNL2_16S	6RD2_16S	6RD2_ITS	6GTL1_16S	2TRNL1_16S_2	04.0002 pM Std	0.0002 pM Std
C	6LVD2_ITS	6LVD2_16S	2TRNL3_16S	2TRNL3_TRNL	2TRNL2_TRNL	2TRNL2_16S	6RD2_16S	6RD2_ITS	6GTL1_16S	2TRNL1_16S_2	0.002 pM Std	0.002 pM Std
D	6LVD2_ITS	6LVD2_16S	2TRNL3_16S	2TRNL3_TRNL	2TRNL2_TRNL	2TRNL2_16S	6RD2_16S	6RD2_ITS	6GTL1_16S	2TRNL1_16S_2	0.02 pM Std	0.02 pM Std
E	6LVD2_ITS	6LVD2_16S	2TRNL3_16S	2TRNL3_TRNL	2TRNL2_TRNL	2TRNL2_16S	6RD2_16S	6RD2_ITS	6GTL1_16S	2TRNL1_16S_2	0.2 pM Std	0.2 pM Std
F	6LVD2_ITS	6LVD2_16S	2TRNL3_16S	2TRNL3_TRNL	2TRNL2_TRNL	2TRNL2_16S	6RD2_16S	6RD2_ITS	6GTL1_16S	2TRNL1_16S_2	2 pM Std	2 pM Std
G	6LVD2_ITS	6LVD2_16S	2TRNL3_16S	2TRNL3_TRNL	2TRNL2_TRNL	2TRNL2_16S	6RD2_16S	6RD2_ITS	6GTL1_16S	2TRNL1_16S_2	20 pM Std	20 pM Std
H	6LVD2_ITS	6LVD2_16S	2TRNL3_16S	2TRNL3_TRNL	2TRNL2_TRNL	2TRNL2_16S	6RD2_16S	6RD2_ITS	6GTL1_16S	2TRNL1_16S_2

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	6LVD2_ITS	6LVD2_16S	2TRNL3_16S	2TRNL3_TRNL	2TRNL2_TRNL	2TRNL2_16S	6RD2_16S	6RD2_ITS	6GTL1_16S	2TRNL1_16S_2	20 pM Std	20 pM Std
B	6LVD2_ITS	6LVD2_16S	2TRNL3_16S	2TRNL3_TRNL	2TRNL2_TRNL	2TRNL2_16S	6RD2_16S	6RD2_ITS	6GTL1_16S	2TRNL1_16S_2	2 pM Std	2 pM Std
C	6LVD2_ITS	6LVD2_16S	2TRNL3_16S	2TRNL3_TRNL	2TRNL2_TRNL	2TRNL2_16S	6RD2_16S	6RD2_ITS	6GTL1_16S	2TRNL1_16S_2	0.2 pM Std	0.2 pM Std
D	6LVD2_ITS	6LVD2_16S	2TRNL3_16S	2TRNL3_TRNL	2TRNL2_TRNL	2TRNL2_16S	6RD2_16S	6RD2_ITS	6GTL1_16S	2TRNL1_16S_2	0.02 pM Std	0.02 pM Std
E	6LVD2_ITS	6LVD2_16S	2TRNL3_16S	2TRNL3_TRNL	2TRNL2_TRNL	2TRNL2_16S	6RD2_16S	6RD2_ITS	6GTL1_16S	2TRNL1_16S_2	0.002 pM Std	0.002 pM Std
F	6LVD2_ITS	6LVD2_16S	2TRNL3_16S	2TRNL3_TRNL	2TRNL2_TRNL	2TRNL2_16S	6RD2_16S	6RD2_ITS	6GTL1_16S	2TRNL1_16S_2	0.0002 pM Std	0.0002 pM Std
G	6LVD2_ITS	6LVD2_16S	2TRNL3_16S	2TRNL3_TRNL	2TRNL2_TRNL	2TRNL2_16S	6RD2_16S	6RD2_ITS	6GTL1_16S	2TRNL1_16S_2	NTC	NTC
H	6LVD2_ITS	6LVD2_16S	2TRNL3_16S	2TRNL3_TRNL	2TRNL2_TRNL	2TRNL2_16S	6RD2_16S	6RD2_ITS	6GTL1_16S	2TRNL1_16S_2	NTC	NTC

Results:

Plate Name	Average Result (nanomoles)

Plate Name	Average Result (nanomoles)
6LVD2_ITS	16.09
6LVD2_16S	21.71
2TRNL3_16S	42.69
2TRNL3_TRNL	8.68
2TRNL2_TRNL	7.51
2TRNL2_16S	36.27
6RD2_16S	28.48
6RD2_ITS	29.45
6GTL1_16S	8.2
2TRNL1_16S_2	32.72

Full qPCR results below:

qPCR NovaSeq6_Set4

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	9	11	12
A	6RD4_16S_Col3	6RD4_ITS_Col3	6RD5_16S_Col3	6RD5_ITS_Col3	6RD6_16S_Col3	6RD6_ITS_Col3	6RD7_16S_Col3	6RD7_ITS_Col3	6LVD5_16S_A1		NTC
B	6RD4_16S_Col3	6RD4_ITS_Col3	6RD5_16S_Col3	6RD5_ITS_Col3	6RD6_16S_Col3	6RD6_ITS_Col3	6RD7_16S_Col3	6RD7_ITS_Col3	6LVD5_16S_B1	04.0002 pM Std	0.0002 pM Std
C	6RD4_16S_Col3	6RD4_ITS_Col3	6RD5_16S_Col3	6RD5_ITS_Col3	6RD6_16S_Col3	6RD6_ITS_Col3	6RD7_16S_Col3	6RD7_ITS_Col3	6LVD5_16S_C1	0.002 pM Std	0.002 pM Std
D	6RD4_16S_Col3	6RD4_ITS_Col3	6RD5_16S_Col3	6RD5_ITS_Col3	6RD6_16S_Col3	6RD6_ITS_Col3	6RD7_16S_Col3	6RD7_ITS_Col3	6LVD5_16S_D1	0.02 pM Std	0.02 pM Std
E	6RD4_16S_Col3	6RD4_ITS_Col3	6RD5_16S_Col3	6RD5_ITS_Col3	6RD6_16S_Col3	6RD6_ITS_Col3	6RD7_16S_Col3	6RD7_ITS_Col3	6LVD5_ITS_A8	0.2 pM Std	0.2 pM Std
F	6RD4_16S_Col3	6RD4_ITS_Col3	6RD5_16S_Col3	6RD5_ITS_Col3	6RD6_16S_Col3	6RD6_ITS_Col3	6RD7_16S_Col3	6RD7_ITS_Col3	6LVD5_ITS_B8	2 pM Std	2 pM Std
G	6RD4_16S_Col3	6RD4_ITS_Col3	6RD5_16S_Col3	6RD5_ITS_Col3	6RD6_16S_Col3	6RD6_ITS_Col3	6RD7_16S_Col3	6RD7_ITS_Col3	6LVD5_ITS_C8	20 pM Std	20 pM Std
H	6RD4_16S_Col3	6RD4_ITS_Col3	6RD5_16S_Col3	6RD5_ITS_Col3	6RD6_16S_Col3	6RD6_ITS_Col3	6RD7_16S_Col3	6RD7_ITS_Col3	6LVD5_ITS_D8

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	6RD4_16S	6RD4_ITS	6RD5_16S	6RD5_ITS	6RD6_16S	6RD6_ITS	6RD7_16S	6RD7_ITS	6LVD5_16S	NTC	NTC	NTC
B	6RD4_16S	6RD4_ITS	6RD5_16S	6RD5_ITS	6RD6_16S	6RD6_ITS	6RD7_16S	6RD7_ITS	6LVD5_16S	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	6RD4_16S	6RD4_ITS	6RD5_16S	6RD5_ITS	6RD6_16S	6RD6_ITS	6RD7_16S	6RD7_ITS	6LVD5_16S	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	6RD4_16S	6RD4_ITS	6RD5_16S	6RD5_ITS	6RD6_16S	6RD6_ITS	6RD7_16S	6RD7_ITS	6LVD5_16S	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	6RD4_16S	6RD4_ITS	6RD5_16S	6RD5_ITS	6RD6_16S	6RD6_ITS	6RD7_16S	6RD7_ITS	6LVD5_ITS	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	6RD4_16S	6RD4_ITS	6RD5_16S	6RD5_ITS	6RD6_16S	6RD6_ITS	6RD7_16S	6RD7_ITS	6LVD5_ITS	2 pM Std	2 pM Std	2 pM Std
G	6RD4_16S	6RD4_ITS	6RD5_16S	6RD5_ITS	6RD6_16S	6RD6_ITS	6RD7_16S	6RD7_ITS	6LVD5_ITS	20 pM Std	20 pM Std	20 pM Std
H	6RD4_16S	6RD4_ITS	6RD5_16S	6RD5_ITS	6RD6_16S	6RD6_ITS	6RD7_16S	6RD7_ITS	6LVD5_ITS	NS6_Pool_1:1000	NS6_Pool_1:1000	NS6_Pool_1:1000

Results:

Plate Name	Average Result (nanomoles)

Plate Name	Average Result (nanomoles)
6RD4_16S	70.66
6RD4_ITS	22.2
6RD5_16S	76.68
6RD5_ITS	86.82
6RD6_16S	32.57
6RD6_ITS	45.66
6RD7_16S	45.71
6RD7_ITS	67.98
6LVD5_16S	50.03
6LVD5_ITS	14.98
NS6_Pool	12.17

Full qPCR results below: