NovaSeq5 1-Step PCR and qPCR

NS5 Plates

5AL1	5AL2	5AL3	5AL4	5AL5
5LVD1	5LVD2	5LVD3	5LVD4	5LVD5 (Add MC to Col 6)
5FB1	5FB2	5FB3	5FB4	5FB5
5DG1	5DG2	5DG3	5DG4	5DG5
5RD1 (2-Step repeat) - click here for the protocol page.
Yoon Plate will be re-pooled

Current total number of plates for ITS/16S PCR: 20 + 1 plate repeat (sm19-fire)

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

Plate	16S Primer	ITS Primer
5AL1	16S0A1	ITS0A1
5AL1	16S0B1	ITS0B1
5AL2	16S0C1	ITS0C1
5AL2	16S0D1	ITS0D1
5AL3	16S0E1	ITS0E1
5AL3	16S0F1	ITS0F1
5AL4	16S0G1	ITS0G1
5AL4	16S0H1	ITS0H1

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)
Store in -20 until ready to qPCR

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	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
5AL5	16S0A2	ITS0A2
5AL5	16S0B2	ITS0B2
5AL6	16S0C2	ITS0C2
5AL6	16S0D2	ITS0D2
5AL7	16S0E2	ITS0E2
5AL7	16S0F2	ITS0F2
5AL8	16S0G2	ITS0G2
5AL8	16S0H2	ITS0H2

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)
Store in -20 until ready to qPCR.

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	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
5LVD1	16S0A3	ITS0A3
5LVD1	16S0B3	ITS0B3
5LVD2	16S0C3	ITS0C3
5LVD2	16S0D3	ITS0D3
5LVD3	16S0E3	ITS0E3
5LVD3	16S0F3	ITS0F3
5LVD4	16S0G3	ITS0G3
5LVD4	16S0H3	ITS0H3

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)
Store in -20 until ready to qPCR.

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	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
5LVD5	16S0A4	ITS0A4
5LVD5	16S0B4	ITS0B4
5FB1	16S0C4	ITS0C4
5FB1	16S0D4	ITS0D4
5FB2	16S0E4	ITS0E4
5FB2	16S0F4	ITS0F4
5FB3	16S0G4	ITS0G4
5FB3	16S0H4	ITS0H4

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)
Store in -20 until ready to qPCR.

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	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
5FB4	16S0A5	ITS0A5
5FB4	16S0B5	ITS0B5
5FB5	16S0C5	ITS0C5
5FB5	16S0D5	ITS0D5
5DG1	16S0E5	ITS0E5
5DG1	16S0F5	ITS0F5
5DG2	16S0G5	ITS0G5
5DG2	16S0H5	ITS0H5

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)
Store in -20 until ready to qPCR.

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	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
5DG3	16S0A6	ITS0A6
5DG3	16S0B6	ITS0B6
5DG4	16S0C6	ITS0C6
5DG4	16S0D6	ITS0D6
5DG5 (used only one MID plate since there were only 6 columns of samples. Replicate done on a single plate).	16S0F6`	ITS0F6

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)
Store in -20 until ready to qPCR.

qPCR NovaSeq5_Set1

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	5AL1_16S_Col3	5AL2_16S_Col3	5AL3_16S_Col3	5AL4_16S_Col3	5AL5_16S_Col3	5AL6_16S_Col3	5AL7_16S_Col3	5AL8_16S_Col3	5AL1_ITS_Col3	NTC	NTC	NTC
B	5AL1_16S_Col3	5AL2_16S_Col3	5AL3_16S_Col3	5AL4_16S_Col3	5AL5_16S_Col3	5AL6_16S_Col3	5AL7_16S_Col3	5AL8_16S_Col3	5AL1_ITS_Col3	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	5AL1_16S_Col3	5AL2_16S_Col3	5AL3_16S_Col3	5AL4_16S_Col3	5AL5_16S_Col3	5AL6_16S_Col3	5AL7_16S_Col3	5AL8_16S_Col3	5AL1_ITS_Col3	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	5AL1_16S_Col3	5AL2_16S_Col3	5AL3_16S_Col3	5AL4_16S_Col3	5AL5_16S_Col3	5AL6_16S_Col3	5AL7_16S_Col3	5AL8_16S_Col3	5AL1_ITS_Col3	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	5AL1_16S_Col3	5AL2_16S_Col3	5AL3_16S_Col3	5AL4_16S_Col3	5AL5_16S_Col3	5AL6_16S_Col3	5AL7_16S_Col3	5AL8_16S_Col3	5AL1_ITS_Col3	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	5AL1_16S_Col3	5AL2_16S_Col3	5AL3_16S_Col3	5AL4_16S_Col3	5AL5_16S_Col3	5AL6_16S_Col3	5AL7_16S_Col3	5AL8_16S_Col3	5AL1_ITS_Col3	2 pM Std	2 pM Std	2 pM Std
G	5AL1_16S_Col3	5AL2_16S_Col3	5AL3_16S_Col3	5AL4_16S_Col3	5AL5_16S_Col3	5AL6_16S_Col3	5AL7_16S_Col3	5AL8_16S_Col3	5AL1_ITS_Col3	20 pM Std	20 pM Std	20 pM Std
H	5AL1_16S_Col3	5AL2_16S_Col3	5AL3_16S_Col3	5AL4_16S_Col3	5AL5_16S_Col3	5AL6_16S_Col3	5AL7_16S_Col3	5AL8_16S_Col3	5AL1_ITS_Col3

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)	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
A	5AL1_16S_Col3	5AL2_16S_Col3	5AL3_16S_Col3	5AL4_16S_Col3	5AL5_16S_Col3	5AL6_16S_Col3	5AL7_16S_Col3	5AL8_16S_Col3	5AL1_ITS_Col3	NTC	NTC	NTC
B	5AL1_16S_Col3	5AL2_16S_Col3	5AL3_16S_Col3	5AL4_16S_Col3	5AL5_16S_Col3	5AL6_16S_Col3	5AL7_16S_Col3	5AL8_16S_Col3	5AL1_ITS_Col3	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	5AL1_16S_Col3	5AL2_16S_Col3	5AL3_16S_Col3	5AL4_16S_Col3	5AL5_16S_Col3	5AL6_16S_Col3	5AL7_16S_Col3	5AL8_16S_Col3	5AL1_ITS_Col3	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	5AL1_16S_Col3	5AL2_16S_Col3	5AL3_16S_Col3	5AL4_16S_Col3	5AL5_16S_Col3	5AL6_16S_Col3	5AL7_16S_Col3	5AL8_16S_Col3	5AL1_ITS_Col3	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	5AL1_16S_Col3	5AL2_16S_Col3	5AL3_16S_Col3	5AL4_16S_Col3	5AL5_16S_Col3	5AL6_16S_Col3	5AL7_16S_Col3	5AL8_16S_Col3	5AL1_ITS_Col3	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	5AL1_16S_Col3	5AL2_16S_Col3	5AL3_16S_Col3	5AL4_16S_Col3	5AL5_16S_Col3	5AL6_16S_Col3	5AL7_16S_Col3	5AL8_16S_Col3	5AL1_ITS_Col3	2 pM Std	2 pM Std	2 pM Std
G	5AL1_16S_Col3	5AL2_16S_Col3	5AL3_16S_Col3	5AL4_16S_Col3	5AL5_16S_Col3	5AL6_16S_Col3	5AL7_16S_Col3	5AL8_16S_Col3	5AL1_ITS_Col3	20 pM Std	20 pM Std	20 pM Std
H	5AL1_16S_Col3	5AL2_16S_Col3	5AL3_16S_Col3	5AL4_16S_Col3	5AL5_16S_Col3	5AL6_16S_Col3	5AL7_16S_Col3	5AL8_16S_Col3	5AL1_ITS_Col3

Results:

Plate Name	Average Result (nanomoles)
5AL1_16S_Col3	15.27
5AL2_16S_Col3	8.38
5AL3_16S_Col3	20.55
5AL4_16S_Col3	59.48
5AL5_16S_Col3	18.49
5AL6_16S_Col3	1.33
5AL7_16S_Col3	10.39
5AL8_16S_Col3	11.29
5AL1_ITS_Col3	68.89

Full qPCR results below:

qPCR NovaSeq5_Set2

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	11	12
A	5AL2_ITS_Col3	5AL3_ITS_Col3	5AL4_ITS_Col3	5AL5_ITS_Col3	5AL6_ITS_Col3	5AL7_ITS_Col3	5AL8_ITS_Col3	5LVD1_16S_Col3	5LVD2_16S_Col3		NTC
B	5AL2_ITS_Col3	5AL3_ITS_Col3	5AL4_ITS_Col3	5AL5_ITS_Col3	5AL6_ITS_Col3	5AL7_ITS_Col3	5AL8_ITS_Col3	5LVD1_16S_Col3	5LVD2_16S_Col3	04.0002 pM Std	0.0002 pM Std
C	5AL2_ITS_Col3	5AL3_ITS_Col3	5AL4_ITS_Col3	5AL5_ITS_Col3	5AL6_ITS_Col3	5AL7_ITS_Col3	5AL8_ITS_Col3	5LVD1_16S_Col3	5LVD2_16S_Col3	0.002 pM Std	0.002 pM Std
D	5AL2_ITS_Col3	5AL3_ITS_Col3	5AL4_ITS_Col3	5AL5_ITS_Col3	5AL6_ITS_Col3	5AL7_ITS_Col3	5AL8_ITS_Col3	5LVD1_16S_Col3	5LVD2_16S_Col3	0.02 pM Std	0.02 pM Std
E	5AL2_ITS_Col3	5AL3_ITS_Col3	5AL4_ITS_Col3	5AL5_ITS_Col3	5AL6_ITS_Col3	5AL7_ITS_Col3	5AL8_ITS_Col3	5LVD1_16S_Col3	5LVD2_16S_Col3	0.2 pM Std	0.2 pM Std
F	5AL2_ITS_Col3	5AL3_ITS_Col3	5AL4_ITS_Col3	5AL5_ITS_Col3	5AL6_ITS_Col3	5AL7_ITS_Col3	5AL8_ITS_Col3	5LVD1_16S_Col3	5LVD2_16S_Col3	2 pM Std	2 pM Std
G	5AL2_ITS_Col3	5AL3_ITS_Col3	5AL4_ITS_Col3	5AL5_ITS_Col3	5AL6_ITS_Col3	5AL7_ITS_Col3	5AL8_ITS_Col3	5LVD1_16S_Col3	5LVD2_16S_Col3	20 pM Std	20 pM Std
H	5AL2_ITS_Col3	5AL3_ITS_Col3	5AL4_ITS_Col3	5AL5_ITS_Col3	5AL6_ITS_Col3	5AL7_ITS_Col3	5AL8_ITS_Col3	5LVD1_16S_Col3	5LVD2_16S_Col3

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)	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
A	5AL2_ITS_Col3	5AL3_ITS_Col3	5AL4_ITS_Col3	5AL5_ITS_Col3	5AL6_ITS_Col3	5AL7_ITS_Col3	5AL8_ITS_Col3	5LVD1_16S_Col3	5LVD2_16S_Col3	NTC	NTC	NTC
B	5AL2_ITS_Col3	5AL3_ITS_Col3	5AL4_ITS_Col3	5AL5_ITS_Col3	5AL6_ITS_Col3	5AL7_ITS_Col3	5AL8_ITS_Col3	5LVD1_16S_Col3	5LVD2_16S_Col3	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	5AL2_ITS_Col3	5AL3_ITS_Col3	5AL4_ITS_Col3	5AL5_ITS_Col3	5AL6_ITS_Col3	5AL7_ITS_Col3	5AL8_ITS_Col3	5LVD1_16S_Col3	5LVD2_16S_Col3	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	5AL2_ITS_Col3	5AL3_ITS_Col3	5AL4_ITS_Col3	5AL5_ITS_Col3	5AL6_ITS_Col3	5AL7_ITS_Col3	5AL8_ITS_Col3	5LVD1_16S_Col3	5LVD2_16S_Col3	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	5AL2_ITS_Col3	5AL3_ITS_Col3	5AL4_ITS_Col3	5AL5_ITS_Col3	5AL6_ITS_Col3	5AL7_ITS_Col3	5AL8_ITS_Col3	5LVD1_16S_Col3	5LVD2_16S_Col3	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	5AL2_ITS_Col3	5AL3_ITS_Col3	5AL4_ITS_Col3	5AL5_ITS_Col3	5AL6_ITS_Col3	5AL7_ITS_Col3	5AL8_ITS_Col3	5LVD1_16S_Col3	5LVD2_16S_Col3	2 pM Std	2 pM Std	2 pM Std
G	5AL2_ITS_Col3	5AL3_ITS_Col3	5AL4_ITS_Col3	5AL5_ITS_Col3	5AL6_ITS_Col3	5AL7_ITS_Col3	5AL8_ITS_Col3	5LVD1_16S_Col3	5LVD2_16S_Col3	20 pM Std	20 pM Std	20 pM Std
H	5AL2_ITS_Col3	5AL3_ITS_Col3	5AL4_ITS_Col3	5AL5_ITS_Col3	5AL6_ITS_Col3	5AL7_ITS_Col3	5AL8_ITS_Col3	5LVD1_16S_Col3	5LVD2_16S_Col3

Results:

Plate Name	Average Result (nanomoles)
5AL2_ITS_Col3	23.49
5AL3_ITS_Col3	25.91
5AL4_ITS_Col3	28.89
5AL5_ITS_Col3	37.02
5AL6_ITS_Col3	18.74
5AL7_ITS_Col3	30.43
5AL8_ITS_Col3	14.8
5LVD1_16S_Col3	61.73
5LVD2_16S_Col3	57.63

Full qPCR results below:

qPCR NovaSeq5_Set3

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	11	12
A	5LVD3_16S_Col3	5LVD4_16S_Col3	5LVD5_16S_Col3	5LVD1_ITS_Col3	5LVD2_ITS_Col3	5LVD3_ITS_Col3	5LVD4_ITS_Col3	5LVD5_ITS_Col3	5FB1_16S_Col3		NTC
B	5LVD3_16S_Col3	5LVD4_16S_Col3	5LVD5_16S_Col3	5LVD1_ITS_Col3	5LVD2_ITS_Col3	5LVD3_ITS_Col3	5LVD4_ITS_Col3	5LVD5_ITS_Col3	5FB1_16S_Col3	04.0002 pM Std	0.0002 pM Std
C	5LVD3_16S_Col3	5LVD4_16S_Col3	5LVD5_16S_Col3	5LVD1_ITS_Col3	5LVD2_ITS_Col3	5LVD3_ITS_Col3	5LVD4_ITS_Col3	5LVD5_ITS_Col3	5FB1_16S_Col3	0.002 pM Std	0.002 pM Std
D	5LVD3_16S_Col3	5LVD4_16S_Col3	5LVD5_16S_Col3	5LVD1_ITS_Col3	5LVD2_ITS_Col3	5LVD3_ITS_Col3	5LVD4_ITS_Col3	5LVD5_ITS_Col3	5FB1_16S_Col3	0.02 pM Std	0.02 pM Std
E	5LVD3_16S_Col3	5LVD4_16S_Col3	5LVD5_16S_Col3	5LVD1_ITS_Col3	5LVD2_ITS_Col3	5LVD3_ITS_Col3	5LVD4_ITS_Col3	5LVD5_ITS_Col3	5FB1_16S_Col3	0.2 pM Std	0.2 pM Std
F	5LVD3_16S_Col3	5LVD4_16S_Col3	5LVD5_16S_Col3	5LVD1_ITS_Col3	5LVD2_ITS_Col3	5LVD3_ITS_Col3	5LVD4_ITS_Col3	5LVD5_ITS_Col3	5FB1_16S_Col3	2 pM Std	2 pM Std
G	5LVD3_16S_Col3	5LVD4_16S_Col3	5LVD5_16S_Col3	5LVD1_ITS_Col3	5LVD2_ITS_Col3	5LVD3_ITS_Col3	5LVD4_ITS_Col3	5LVD5_ITS_Col3	5FB1_16S_Col3	20 pM Std	20 pM Std
H	5LVD3_16S_Col3	5LVD4_16S_Col3	5LVD5_16S_Col3	5LVD1_ITS_Col3	5LVD2_ITS_Col3	5LVD3_ITS_Col3	5LVD4_ITS_Col3	5LVD5_ITS_Col3	5FB1_16S_Col3

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)	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
A	5LVD3_16S_Col3	5LVD4_16S_Col3	5LVD5_16S_Col3	5LVD1_ITS_Col3	5LVD2_ITS_Col3	5LVD3_ITS_Col3	5LVD4_ITS_Col3	5LVD5_ITS_Col3	5FB1_16S_Col3	NTC	NTC	NTC
B	5LVD3_16S_Col3	5LVD4_16S_Col3	5LVD5_16S_Col3	5LVD1_ITS_Col3	5LVD2_ITS_Col3	5LVD3_ITS_Col3	5LVD4_ITS_Col3	5LVD5_ITS_Col3	5FB1_16S_Col3	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	5LVD3_16S_Col3	5LVD4_16S_Col3	5LVD5_16S_Col3	5LVD1_ITS_Col3	5LVD2_ITS_Col3	5LVD3_ITS_Col3	5LVD4_ITS_Col3	5LVD5_ITS_Col3	5FB1_16S_Col3	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	5LVD3_16S_Col3	5LVD4_16S_Col3	5LVD5_16S_Col3	5LVD1_ITS_Col3	5LVD2_ITS_Col3	5LVD3_ITS_Col3	5LVD4_ITS_Col3	5LVD5_ITS_Col3	5FB1_16S_Col3	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	5LVD3_16S_Col3	5LVD4_16S_Col3	5LVD5_16S_Col3	5LVD1_ITS_Col3	5LVD2_ITS_Col3	5LVD3_ITS_Col3	5LVD4_ITS_Col3	5LVD5_ITS_Col3	5FB1_16S_Col3	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	5LVD3_16S_Col3	5LVD4_16S_Col3	5LVD5_16S_Col3	5LVD1_ITS_Col3	5LVD2_ITS_Col3	5LVD3_ITS_Col3	5LVD4_ITS_Col3	5LVD5_ITS_Col3	5FB1_16S_Col3	2 pM Std	2 pM Std	2 pM Std
G	5LVD3_16S_Col3	5LVD4_16S_Col3	5LVD5_16S_Col3	5LVD1_ITS_Col3	5LVD2_ITS_Col3	5LVD3_ITS_Col3	5LVD4_ITS_Col3	5LVD5_ITS_Col3	5FB1_16S_Col3	20 pM Std	20 pM Std	20 pM Std
H	5LVD3_16S_Col3	5LVD4_16S_Col3	5LVD5_16S_Col3	5LVD1_ITS_Col3	5LVD2_ITS_Col3	5LVD3_ITS_Col3	5LVD4_ITS_Col3	5LVD5_ITS_Col3	5FB1_16S_Col3

Results:

Plate Name	Average Result (nanomoles)
5LVD3_16S_Col3	60.49
5LVD4_16S_Col3	56.58
5LVD5_16S_Col3	59.58
5LVD1_ITS_Col3	52.72
5LVD2_ITS_Col3	25.43
5LVD3_ITS_Col3	39.91
5LVD4_ITS_Col3	38.61
5LVD5_ITS_Col3	21.75
5FB1_16S_Col3	66.78

Full qPCR results below:

qPCR NovaSeq5_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	11	12
A	5FB2_16S_Col3	5FB3_16S_Col3	5FB4_16S_Col3	5FB5_16S_Col3	5FB1_ITS_Col3	5FB2_ITS_Col3	5FB3_ITS_Col3	5FB4_ITS_Col3	5FB5_ITS_Col3		NTC
B	5FB2_16S_Col3	5FB3_16S_Col3	5FB4_16S_Col3	5FB5_16S_Col3	5FB1_ITS_Col3	5FB2_ITS_Col3	5FB3_ITS_Col3	5FB4_ITS_Col3	5FB5_ITS_Col3	04.0002 pM Std	0.0002 pM Std
C	5FB2_16S_Col3	5FB3_16S_Col3	5FB4_16S_Col3	5FB5_16S_Col3	5FB1_ITS_Col3	5FB2_ITS_Col3	5FB3_ITS_Col3	5FB4_ITS_Col3	5FB5_ITS_Col3	0.002 pM Std	0.002 pM Std
D	5FB2_16S_Col3	5FB3_16S_Col3	5FB4_16S_Col3	5FB5_16S_Col3	5FB1_ITS_Col3	5FB2_ITS_Col3	5FB3_ITS_Col3	5FB4_ITS_Col3	5FB5_ITS_Col3	0.02 pM Std	0.02 pM Std
E	5FB2_16S_Col3	5FB3_16S_Col3	5FB4_16S_Col3	5FB5_16S_Col3	5FB1_ITS_Col3	5FB2_ITS_Col3	5FB3_ITS_Col3	5FB4_ITS_Col3	5FB5_ITS_Col3	0.2 pM Std	0.2 pM Std
F	5FB2_16S_Col3	5FB3_16S_Col3	5FB4_16S_Col3	5FB5_16S_Col3	5FB1_ITS_Col3	5FB2_ITS_Col3	5FB3_ITS_Col3	5FB4_ITS_Col3	5FB5_ITS_Col3	2 pM Std	2 pM Std
G	5FB2_16S_Col3	5FB3_16S_Col3	5FB4_16S_Col3	5FB5_16S_Col3	5FB1_ITS_Col3	5FB2_ITS_Col3	5FB3_ITS_Col3	5FB4_ITS_Col3	5FB5_ITS_Col3	20 pM Std	20 pM Std
H	5FB2_16S_Col3	5FB3_16S_Col3	5FB4_16S_Col3	5FB5_16S_Col3	5FB1_ITS_Col3	5FB2_ITS_Col3	5FB3_ITS_Col3	5FB4_ITS_Col3	5FB5_ITS_Col3

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)	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
A	5FB2_16S_Col3	5FB3_16S_Col3	5FB4_16S_Col3	5FB5_16S_Col3	5FB1_ITS_Col3	5FB2_ITS_Col3	5FB3_ITS_Col3	5FB4_ITS_Col3	5FB5_ITS_Col3	NTC	NTC	NTC
B	5FB2_16S_Col3	5FB3_16S_Col3	5FB4_16S_Col3	5FB5_16S_Col3	5FB1_ITS_Col3	5FB2_ITS_Col3	5FB3_ITS_Col3	5FB4_ITS_Col3	5FB5_ITS_Col3	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	5FB2_16S_Col3	5FB3_16S_Col3	5FB4_16S_Col3	5FB5_16S_Col3	5FB1_ITS_Col3	5FB2_ITS_Col3	5FB3_ITS_Col3	5FB4_ITS_Col3	5FB5_ITS_Col3	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	5FB2_16S_Col3	5FB3_16S_Col3	5FB4_16S_Col3	5FB5_16S_Col3	5FB1_ITS_Col3	5FB2_ITS_Col3	5FB3_ITS_Col3	5FB4_ITS_Col3	5FB5_ITS_Col3	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	5FB2_16S_Col3	5FB3_16S_Col3	5FB4_16S_Col3	5FB5_16S_Col3	5FB1_ITS_Col3	5FB2_ITS_Col3	5FB3_ITS_Col3	5FB4_ITS_Col3	5FB5_ITS_Col3	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	5FB2_16S_Col3	5FB3_16S_Col3	5FB4_16S_Col3	5FB5_16S_Col3	5FB1_ITS_Col3	5FB2_ITS_Col3	5FB3_ITS_Col3	5FB4_ITS_Col3	5FB5_ITS_Col3	2 pM Std	2 pM Std	2 pM Std
G	5FB2_16S_Col3	5FB3_16S_Col3	5FB4_16S_Col3	5FB5_16S_Col3	5FB1_ITS_Col3	5FB2_ITS_Col3	5FB3_ITS_Col3	5FB4_ITS_Col3	5FB5_ITS_Col3	20 pM Std	20 pM Std	20 pM Std
H	5FB2_16S_Col3	5FB3_16S_Col3	5FB4_16S_Col3	5FB5_16S_Col3	5FB1_ITS_Col3	5FB2_ITS_Col3	5FB3_ITS_Col3	5FB4_ITS_Col3	5FB5_ITS_Col3

Results:

Plate Name	Average Result (nanomoles)
5FB2_16S_Col3	35.64
5FB3_16S_Col3	31.55
5FB4_16S_Col3	45.58
5FB5_16S_Col3	9.5
5FB1_ITS_Col3	19.46
5FB2_ITS_Col3	83.23
5FB3_ITS_Col3	59.86
5FB4_ITS_Col3	45.49
5FB5_ITS_Col3	16.69

Full qPCR results below:

qPCR NovaSeq5_Set5

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	11	12
A	5DG1_16S_Col3	5DG2_16S_Col3	5DG3_16S_Col3	5DG4_16S_Col3	5DG5_16S_Col3	5DG1_ITS_Col3	5DG2_ITS_Col3	5DG3_ITS_Col3	5DG4_ITS_Col3		NTC
B	5DG1_16S_Col3	5DG2_16S_Col3	5DG3_16S_Col3	5DG4_16S_Col3	5DG5_16S_Col3	5DG1_ITS_Col3	5DG2_ITS_Col3	5DG3_ITS_Col3	5DG4_ITS_Col3	04.0002 pM Std	0.0002 pM Std
C	5DG1_16S_Col3	5DG2_16S_Col3	5DG3_16S_Col3	5DG4_16S_Col3	5DG5_16S_Col3	5DG1_ITS_Col3	5DG2_ITS_Col3	5DG3_ITS_Col3	5DG4_ITS_Col3	0.002 pM Std	0.002 pM Std
D	5DG1_16S_Col3	5DG2_16S_Col3	5DG3_16S_Col3	5DG4_16S_Col3	5DG5_16S_Col3	5DG1_ITS_Col3	5DG2_ITS_Col3	5DG3_ITS_Col3	5DG4_ITS_Col3	0.02 pM Std	0.02 pM Std
E	5DG1_16S_Col3	5DG2_16S_Col3	5DG3_16S_Col3	5DG4_16S_Col3	5DG5_16S_Col3	5DG1_ITS_Col3	5DG2_ITS_Col3	5DG3_ITS_Col3	5DG4_ITS_Col3	0.2 pM Std	0.2 pM Std
F	5DG1_16S_Col3	5DG2_16S_Col3	5DG3_16S_Col3	5DG4_16S_Col3	5DG5_16S_Col3	5DG1_ITS_Col3	5DG2_ITS_Col3	5DG3_ITS_Col3	5DG4_ITS_Col3	2 pM Std	2 pM Std
G	5DG1_16S_Col3	5DG2_16S_Col3	5DG3_16S_Col3	5DG4_16S_Col3	5DG5_16S_Col3	5DG1_ITS_Col3	5DG2_ITS_Col3	5DG3_ITS_Col3	5DG4_ITS_Col3	20 pM Std	20 pM Std
H	5DG1_16S_Col3	5DG2_16S_Col3	5DG3_16S_Col3	5DG4_16S_Col3	5DG5_16S_Col3	5DG1_ITS_Col3	5DG2_ITS_Col3	5DG3_ITS_Col3	5DG4_ITS_Col3

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)	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
A	5DG1_16S_Col3	5DG2_16S_Col3	5DG3_16S_Col3	5DG4_16S_Col3	5DG5_16S_Col3	5DG1_ITS_Col3	5DG2_ITS_Col3	5DG3_ITS_Col3	5DG4_ITS_Col3	NTC	NTC	NTC
B	5DG1_16S_Col3	5DG2_16S_Col3	5DG3_16S_Col3	5DG4_16S_Col3	5DG5_16S_Col3	5DG1_ITS_Col3	5DG2_ITS_Col3	5DG3_ITS_Col3	5DG4_ITS_Col3	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	5DG1_16S_Col3	5DG2_16S_Col3	5DG3_16S_Col3	5DG4_16S_Col3	5DG5_16S_Col3	5DG1_ITS_Col3	5DG2_ITS_Col3	5DG3_ITS_Col3	5DG4_ITS_Col3	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	5DG1_16S_Col3	5DG2_16S_Col3	5DG3_16S_Col3	5DG4_16S_Col3	5DG5_16S_Col3	5DG1_ITS_Col3	5DG2_ITS_Col3	5DG3_ITS_Col3	5DG4_ITS_Col3	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	5DG1_16S_Col3	5DG2_16S_Col3	5DG3_16S_Col3	5DG4_16S_Col3	5DG5_16S_Col3	5DG1_ITS_Col3	5DG2_ITS_Col3	5DG3_ITS_Col3	5DG4_ITS_Col3	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	5DG1_16S_Col3	5DG2_16S_Col3	5DG3_16S_Col3	5DG4_16S_Col3	5DG5_16S_Col3	5DG1_ITS_Col3	5DG2_ITS_Col3	5DG3_ITS_Col3	5DG4_ITS_Col3	2 pM Std	2 pM Std	2 pM Std
G	5DG1_16S_Col3	5DG2_16S_Col3	5DG3_16S_Col3	5DG4_16S_Col3	5DG5_16S_Col3	5DG1_ITS_Col3	5DG2_ITS_Col3	5DG3_ITS_Col3	5DG4_ITS_Col3	20 pM Std	20 pM Std	20 pM Std
H	5DG1_16S_Col3	5DG2_16S_Col3	5DG3_16S_Col3	5DG4_16S_Col3	5DG5_16S_Col3	5DG1_ITS_Col3	5DG2_ITS_Col3	5DG3_ITS_Col3	5DG4_ITS_Col3

Results:

Plate Name	Average Result (nanomoles)
5DG1_16S_Col3
5DG2_16S_Col3
5DG3_16S_Col3
5DG4_16S_Col3
5DG5_16S_Col3
5DG1_ITS_Col3
5DG2_ITS_Col3
5DG3_ITS_Col3
5DG4_ITS_Col3

Full qPCR results below:

qPCR NovaSeq5_Set6

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	11	12
A	5DG5_ITS_Col3	5RD1_16S_Col1	5RD1_16S_Col2	5RD1_16S_Col3	5RD1_16S_Col4	5RD1_16S_Col5	5RD1_16S_Col6	5RD1_16S_Col7	5RD1_ITS_Col1		NTC
B	5DG5_ITS_Col3	5RD1_16S_Col1	5RD1_16S_Col2	5RD1_16S_Col3	5RD1_16S_Col4	5RD1_16S_Col5	5RD1_16S_Col6	5RD1_16S_Col7	5RD1_ITS_Col1	04.0002 pM Std	0.0002 pM Std
C	5DG5_ITS_Col3	5RD1_16S_Col1	5RD1_16S_Col2	5RD1_16S_Col3	5RD1_16S_Col4	5RD1_16S_Col5	5RD1_16S_Col6	5RD1_16S_Col7	5RD1_ITS_Col1	0.002 pM Std	0.002 pM Std
D	5DG5_ITS_Col3	5RD1_16S_Col1	5RD1_16S_Col2	5RD1_16S_Col3	5RD1_16S_Col4	5RD1_16S_Col5	5RD1_16S_Col6	5RD1_16S_Col7	5RD1_ITS_Col1	0.02 pM Std	0.02 pM Std
E	5DG5_ITS_Col3	5RD1_16S_Col1	5RD1_16S_Col2	5RD1_16S_Col3	5RD1_16S_Col4	5RD1_16S_Col5	5RD1_16S_Col6	5RD1_16S_Col7	5RD1_ITS_Col1	0.2 pM Std	0.2 pM Std
F	5DG5_ITS_Col3	5RD1_16S_Col1	5RD1_16S_Col2	5RD1_16S_Col3	5RD1_16S_Col4	5RD1_16S_Col5	5RD1_16S_Col6	5RD1_16S_Col7	5RD1_ITS_Col1	2 pM Std	2 pM Std
G	5DG5_ITS_Col3	5RD1_16S_Col1	5RD1_16S_Col2	5RD1_16S_Col3	5RD1_16S_Col4	5RD1_16S_Col5	5RD1_16S_Col6	5RD1_16S_Col7	5RD1_ITS_Col1	20 pM Std	20 pM Std
H	5DG5_ITS_Col3	5RD1_16S_Col1	5RD1_16S_Col2	5RD1_16S_Col3	5RD1_16S_Col4	5RD1_16S_Col5	5RD1_16S_Col6	5RD1_16S_Col7	5RD1_ITS_Col1

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)	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
A	5DG5_ITS_Col3	5RD1_16S_Col1	5RD1_16S_Col2	5RD1_16S_Col3	5RD1_16S_Col4	5RD1_16S_Col5	5RD1_16S_Col6	5RD1_16S_Col7	5RD1_ITS_Col1	NTC	NTC	NTC
B	5DG5_ITS_Col3	5RD1_16S_Col1	5RD1_16S_Col2	5RD1_16S_Col3	5RD1_16S_Col4	5RD1_16S_Col5	5RD1_16S_Col6	5RD1_16S_Col7	5RD1_ITS_Col1	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	5DG5_ITS_Col3	5RD1_16S_Col1	5RD1_16S_Col2	5RD1_16S_Col3	5RD1_16S_Col4	5RD1_16S_Col5	5RD1_16S_Col6	5RD1_16S_Col7	5RD1_ITS_Col1	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	5DG5_ITS_Col3	5RD1_16S_Col1	5RD1_16S_Col2	5RD1_16S_Col3	5RD1_16S_Col4	5RD1_16S_Col5	5RD1_16S_Col6	5RD1_16S_Col7	5RD1_ITS_Col1	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	5DG5_ITS_Col3	5RD1_16S_Col1	5RD1_16S_Col2	5RD1_16S_Col3	5RD1_16S_Col4	5RD1_16S_Col5	5RD1_16S_Col6	5RD1_16S_Col7	5RD1_ITS_Col1	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	5DG5_ITS_Col3	5RD1_16S_Col1	5RD1_16S_Col2	5RD1_16S_Col3	5RD1_16S_Col4	5RD1_16S_Col5	5RD1_16S_Col6	5RD1_16S_Col7	5RD1_ITS_Col1	2 pM Std	2 pM Std	2 pM Std
G	5DG5_ITS_Col3	5RD1_16S_Col1	5RD1_16S_Col2	5RD1_16S_Col3	5RD1_16S_Col4	5RD1_16S_Col5	5RD1_16S_Col6	5RD1_16S_Col7	5RD1_ITS_Col1	20 pM Std	20 pM Std	20 pM Std
H	5DG5_ITS_Col3	5RD1_16S_Col1	5RD1_16S_Col2	5RD1_16S_Col3	5RD1_16S_Col4	5RD1_16S_Col5	5RD1_16S_Col6	5RD1_16S_Col7	5RD1_ITS_Col1

Results:

Plate Name	Average Result (nanomoles)
5DG5_ITS_Col3
5RD1_16S_Col1
5RD1_16S_Col2
5RD1_16S_Col3
5RD1_16S_Col4
5RD1_16S_Col5
5RD1_16S_Col6
5RD1_16S_Col7
5RD1_ITS_Col1

Full qPCR results below:

qPCR NovaSeq5_Set7

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	11	12
A	5RD1_ITS_Col2	5RD1_ITS_Col3	5RD1_ITS_Col4	5RD1_ITS_Col5	5RD1_ITS_Col6	5RD1_ITS_Col7	Ha_Yoon_16S_ITS_Col1	Ha_Yoon_16S_ITS_Col6	Ha_Yoon_16S_ITS_Col12		NTC
B	5RD1_ITS_Col2	5RD1_ITS_Col3	5RD1_ITS_Col4	5RD1_ITS_Col5	5RD1_ITS_Col6	5RD1_ITS_Col7	Ha_Yoon_16S_ITS_Col1	Ha_Yoon_16S_ITS_Col6	Ha_Yoon_16S_ITS_Col12	04.0002 pM Std	0.0002 pM Std
C	5RD1_ITS_Col2	5RD1_ITS_Col3	5RD1_ITS_Col4	5RD1_ITS_Col5	5RD1_ITS_Col6	5RD1_ITS_Col7	Ha_Yoon_16S_ITS_Col1	Ha_Yoon_16S_ITS_Col6	Ha_Yoon_16S_ITS_Col12	0.002 pM Std	0.002 pM Std
D	5RD1_ITS_Col2	5RD1_ITS_Col3	5RD1_ITS_Col4	5RD1_ITS_Col5	5RD1_ITS_Col6	5RD1_ITS_Col7	Ha_Yoon_16S_ITS_Col1	Ha_Yoon_16S_ITS_Col6	Ha_Yoon_16S_ITS_Col12	0.02 pM Std	0.02 pM Std
E	5RD1_ITS_Col2	5RD1_ITS_Col3	5RD1_ITS_Col4	5RD1_ITS_Col5	5RD1_ITS_Col6	5RD1_ITS_Col7	Ha_Yoon_16S_ITS_Col1	Ha_Yoon_16S_ITS_Col6	Ha_Yoon_16S_ITS_Col12	0.2 pM Std	0.2 pM Std
F	5RD1_ITS_Col2	5RD1_ITS_Col3	5RD1_ITS_Col4	5RD1_ITS_Col5	5RD1_ITS_Col6	5RD1_ITS_Col7	Ha_Yoon_16S_ITS_Col1	Ha_Yoon_16S_ITS_Col6	Ha_Yoon_16S_ITS_Col12	2 pM Std	2 pM Std
G	5RD1_ITS_Col2	5RD1_ITS_Col3	5RD1_ITS_Col4	5RD1_ITS_Col5	5RD1_ITS_Col6	5RD1_ITS_Col7	Ha_Yoon_16S_ITS_Col1	Ha_Yoon_16S_ITS_Col6	Ha_Yoon_16S_ITS_Col12	20 pM Std	20 pM Std
H	5RD1_ITS_Col2	5RD1_ITS_Col3	5RD1_ITS_Col4	5RD1_ITS_Col5	5RD1_ITS_Col6	5RD1_ITS_Col7	Ha_Yoon_16S_ITS_Col1	Ha_Yoon_16S_ITS_Col6	Ha_Yoon_16S_ITS_Col12

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)	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
A	5RD1_ITS_Col2	5RD1_ITS_Col3	5RD1_ITS_Col4	5RD1_ITS_Col5	5RD1_ITS_Col6	5RD1_ITS_Col7	Ha_Yoon_16S_ITS_Col1	Ha_Yoon_16S_ITS_Col6	Ha_Yoon_16S_ITS_Col12	NTC	NTC	NTC
B	5RD1_ITS_Col2	5RD1_ITS_Col3	5RD1_ITS_Col4	5RD1_ITS_Col5	5RD1_ITS_Col6	5RD1_ITS_Col7	Ha_Yoon_16S_ITS_Col1	Ha_Yoon_16S_ITS_Col6	Ha_Yoon_16S_ITS_Col12	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	5RD1_ITS_Col2	5RD1_ITS_Col3	5RD1_ITS_Col4	5RD1_ITS_Col5	5RD1_ITS_Col6	5RD1_ITS_Col7	Ha_Yoon_16S_ITS_Col1	Ha_Yoon_16S_ITS_Col6	Ha_Yoon_16S_ITS_Col12	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	5RD1_ITS_Col2	5RD1_ITS_Col3	5RD1_ITS_Col4	5RD1_ITS_Col5	5RD1_ITS_Col6	5RD1_ITS_Col7	Ha_Yoon_16S_ITS_Col1	Ha_Yoon_16S_ITS_Col6	Ha_Yoon_16S_ITS_Col12	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	5RD1_ITS_Col2	5RD1_ITS_Col3	5RD1_ITS_Col4	5RD1_ITS_Col5	5RD1_ITS_Col6	5RD1_ITS_Col7	Ha_Yoon_16S_ITS_Col1	Ha_Yoon_16S_ITS_Col6	Ha_Yoon_16S_ITS_Col12	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	5RD1_ITS_Col2	5RD1_ITS_Col3	5RD1_ITS_Col4	5RD1_ITS_Col5	5RD1_ITS_Col6	5RD1_ITS_Col7	Ha_Yoon_16S_ITS_Col1	Ha_Yoon_16S_ITS_Col6	Ha_Yoon_16S_ITS_Col12	2 pM Std	2 pM Std	2 pM Std
G	5RD1_ITS_Col2	5RD1_ITS_Col3	5RD1_ITS_Col4	5RD1_ITS_Col5	5RD1_ITS_Col6	5RD1_ITS_Col7	Ha_Yoon_16S_ITS_Col1	Ha_Yoon_16S_ITS_Col6	Ha_Yoon_16S_ITS_Col12	20 pM Std	20 pM Std	20 pM Std
H	5RD1_ITS_Col2	5RD1_ITS_Col3	5RD1_ITS_Col4	5RD1_ITS_Col5	5RD1_ITS_Col6	5RD1_ITS_Col7	Ha_Yoon_16S_ITS_Col1	Ha_Yoon_16S_ITS_Col6	Ha_Yoon_16S_ITS_Col12

Results:

Plate Name	Average Result (nanomoles)
5RD1_ITS_Col2
5RD1_ITS_Col3
5RD1_ITS_Col4
5RD1_ITS_Col5
5RD1_ITS_Col6
5RD1_ITS_Col7
Ha_Yoon_16S_ITS_Col1
Ha_Yoon_16S_ITS_Col6
Ha_Yoon_16S_ITS_Col12

Full qPCR results below: