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*
5AC1*	5AC2*	5AC3*

*Red plates TBD if they will be on NS5

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 “NovaSeq4 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)

qPCR NovaSeq4_Plate1

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	4AH1_16S	4AH1_ITS	4AH2_16S	4AH2_ITS	4PA1_16S	4PA1_ITS	4PA2_16S	4PA2_ITS	NTC	NTC	NTC
B	4AH1_16S	4AH1_ITS	4AH2_16S	4AH2_ITS	4PA1_16S	4PA1_ITS	4PA2_16S	4PA2_ITS	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	4AH1_16S	4AH1_ITS	4AH2_16S	4AH2_ITS	4PA1_16S	4PA1_ITS	4PA2_16S	4PA2_ITS	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	4AH1_16S	4AH1_ITS	4AH2_16S	4AH2_ITS	4PA1_16S	4PA1_ITS	4PA2_16S	4PA2_ITS	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	4AH1_16S	4AH1_ITS	4AH2_16S	4AH2_ITS	4PA1_16S	4PA1_ITS	4PA2_16S	4PA2_ITS	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	4AH1_16S	4AH1_ITS	4AH2_16S	4AH2_ITS	4PA1_16S	4PA1_ITS	4PA2_16S	4PA2_ITS	2 pM Std	2 pM Std	2 pM Std
G	4AH1_16S	4AH1_ITS	4AH2_16S	4AH2_ITS	4PA1_16S	4PA1_ITS	4PA2_16S	4PA2_ITS	20 pM Std	20 pM Std	20 pM Std
H	4AH1_16S	4AH1_ITS	4AH2_16S	4AH2_ITS	4PA1_16S	4PA1_ITS	4PA2_16S	4PA2_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)	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	10	11	12
A	4AH1_16S	4AH1_ITS	4AH2_16S	4AH2_ITS	4PA1_16S	4PA1_ITS	4PA2_16S	4PA2_ITS	NTC	NTC	NTC
B	4AH1_16S	4AH1_ITS	4AH2_16S	4AH2_ITS	4PA1_16S	4PA1_ITS	4PA2_16S	4PA2_ITS	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	4AH1_16S	4AH1_ITS	4AH2_16S	4AH2_ITS	4PA1_16S	4PA1_ITS	4PA2_16S	4PA2_ITS	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	4AH1_16S	4AH1_ITS	4AH2_16S	4AH2_ITS	4PA1_16S	4PA1_ITS	4PA2_16S	4PA2_ITS	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	4AH1_16S	4AH1_ITS	4AH2_16S	4AH2_ITS	4PA1_16S	4PA1_ITS	4PA2_16S	4PA2_ITS	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	4AH1_16S	4AH1_ITS	4AH2_16S	4AH2_ITS	4PA1_16S	4PA1_ITS	4PA2_16S	4PA2_ITS	2 pM Std	2 pM Std	2 pM Std
G	4AH1_16S	4AH1_ITS	4AH2_16S	4AH2_ITS	4PA1_16S	4PA1_ITS	4PA2_16S	4PA2_ITS	20 pM Std	20 pM Std	20 pM Std
H	4AH1_16S	4AH1_ITS	4AH2_16S	4AH2_ITS	4PA1_16S	4PA1_ITS	4PA2_16S	4PA2_ITS

Results:

Average results for the following plates (column 3):

4AH1_16S: 11.86 nanomoles

4AH1_ITS: 15.74 nanomoles

4AH2_16S: 36.27 nanomoles

4AH2_ITS: 43.5 nanomoles

4PA1_16S: 4.72 nanomoles

4PA1_ITS: 22.76 nanomoles

4PA2_16S: 8.58 nanomoles

4PA2_ITS: 84.66 nanomoles

Results from the first qPCR plate look good. We will continue by checking one pooled plate on the iSeq for further validation. Full qPCR results are 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	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 “NovaSeq4 PCR”. (If doing manually, one needs 2 ul of template and 2 ul of the primers).

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 NovaSeq4_Plate2

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	4PA3_16S	4PA3_ITS	4PA4_16s	4PA4_ITS	4PA5_16S	4PA5_ITS	4PA6_16S	4PA6_ITS		NTC
B	4PA3_16S	4PA3_ITS	4PA4_16s	4PA4_ITS	4PA5_16S	4PA5_ITS	4PA6_16S	4PA6_ITS	04.0002 pM Std	0.0002 pM Std
C	4PA3_16S	4PA3_ITS	4PA4_16s	4PA4_ITS	4PA5_16S	4PA5_ITS	4PA6_16S	4PA6_ITS	0.002 pM Std	0.002 pM Std
D	4PA3_16S	4PA3_ITS	4PA4_16s	4PA4_ITS	4PA5_16S	4PA5_ITS	4PA6_16S	4PA6_ITS	0.02 pM Std	0.02 pM Std
E	4PA3_16S	4PA3_ITS	4PA4_16s	4PA4_ITS	4PA5_16S	4PA5_ITS	4PA6_16S	4PA6_ITS	0.2 pM Std	0.2 pM Std
F	4PA3_16S	4PA3_ITS	4PA4_16s	4PA4_ITS	4PA5_16S	4PA5_ITS	4PA6_16S	4PA6_ITS	2 pM Std	2 pM Std
G	4PA3_16S	4PA3_ITS	4PA4_16s	4PA4_ITS	4PA5_16S	4PA5_ITS	4PA6_16S	4PA6_ITS	20 pM Std	20 pM Std
H	4PA3_16S	4PA3_ITS	4PA4_16s	4PA4_ITS	4PA5_16S	4PA5_ITS	4PA6_16S	4PA6_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	4PA3_16S	4PA3_ITS	4PA4_16s	4PA4_ITS	4PA5_16S	4PA5_ITS	4PA6_16S	4PA6_ITS	NTC	NTC	NTC
B	4PA3_16S	4PA3_ITS	4PA4_16s	4PA4_ITS	4PA5_16S	4PA5_ITS	4PA6_16S	4PA6_ITS	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	4PA3_16S	4PA3_ITS	4PA4_16s	4PA4_ITS	4PA5_16S	4PA5_ITS	4PA6_16S	4PA6_ITS	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	4PA3_16S	4PA3_ITS	4PA4_16s	4PA4_ITS	4PA5_16S	4PA5_ITS	4PA6_16S	4PA6_ITS	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	4PA3_16S	4PA3_ITS	4PA4_16s	4PA4_ITS	4PA5_16S	4PA5_ITS	4PA6_16S	4PA6_ITS	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	4PA3_16S	4PA3_ITS	4PA4_16s	4PA4_ITS	4PA5_16S	4PA5_ITS	4PA6_16S	4PA6_ITS	2 pM Std	2 pM Std	2 pM Std
G	4PA3_16S	4PA3_ITS	4PA4_16s	4PA4_ITS	4PA5_16S	4PA5_ITS	4PA6_16S	4PA6_ITS	20 pM Std	20 pM Std	20 pM Std
H	4PA3_16S	4PA3_ITS	4PA4_16s	4PA4_ITS	4PA5_16S	4PA5_ITS	4PA6_16S	4PA6_ITS

Results:

Average results for the following plates (column 3):

4PA3_16S: 4.04 nanomoles

4PA3_ITS: 46.07 nanomoles

4PA4_16S: 71.0 nanomoles

4PA4_ITS: 68.5 nanomoles

4PA5_16S: 10.26 nanomoles

4PA5_ITS: 41.22 nanomoles

4PA6_16S: 11.57 nanomoles

4PA6_ITS: 44.52 nanomoles

Quantities look good for sequencing. The full result report can be viewed below:

MasterMix (make for 12 plates in 15mL conical tube)

ul/rxn	Reagent	# of rxns	ul needed
3	5X Kapa HiFi Buffer	1210	3630
0.45	10M dNTPs	1210	544.5
0.3	Kapa HiFi HotStart DNA Pol	1210	363
7.25	HPLC H2O	1210	8772.5
11	Total Volume	1210	13310

Add 11 ul to each well of a hard shell, full skirt plate. Seal with bubble strips and store in refrigerator until needed labeled “NovaSeq4 PCR”. (If doing manually, one needs 2 ul of template and 2 ul of the primers).

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 NovaSeq4_Plate3

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	4MR1_16S	4MR1_ITS	4MR2_16S	4MR2_ITS	4SC1_16S	4SC1_ITS	4SC2_16S	4SC2_ITS		NTC
B	4MR1_16S	4MR1_ITS	4MR2_16S	4MR2_ITS	4SC1_16S	4SC1_ITS	4SC2_16S	4SC2_ITS	04.0002 pM Std	0.0002 pM Std
C	4MR1_16S	4MR1_ITS	4MR2_16S	4MR2_ITS	4SC1_16S	4SC1_ITS	4SC2_16S	4SC2_ITS	0.002 pM Std	0.002 pM Std
D	4MR1_16S	4MR1_ITS	4MR2_16S	4MR2_ITS	4SC1_16S	4SC1_ITS	4SC2_16S	4SC2_ITS	0.02 pM Std	0.02 pM Std
E	4MR1_16S	4MR1_ITS	4MR2_16S	4MR2_ITS	4SC1_16S	4SC1_ITS	4SC2_16S	4SC2_ITS	0.2 pM Std	0.2 pM Std
F	4MR1_16S	4MR1_ITS	4MR2_16S	4MR2_ITS	4SC1_16S	4SC1_ITS	4SC2_16S	4SC2_ITS	2 pM Std	2 pM Std
G	4MR1_16S	4MR1_ITS	4MR2_16S	4MR2_ITS	4SC1_16S	4SC1_ITS	4SC2_16S	4SC2_ITS	20 pM Std	20 pM Std
H	4MR1_16S	4MR1_ITS	4MR2_16S	4MR2_ITS	4SC1_16S	4SC1_ITS	4SC2_16S	4SC2_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	4MR1_16S	4MR1_ITS	4MR2_16S	4MR2_ITS	4SC1_16S	4SC1_ITS	4SC2_16S	4SC2_ITS	NTC	NTC	NTC
B	4MR1_16S	4MR1_ITS	4MR2_16S	4MR2_ITS	4SC1_16S	4SC1_ITS	4SC2_16S	4SC2_ITS	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	4MR1_16S	4MR1_ITS	4MR2_16S	4MR2_ITS	4SC1_16S	4SC1_ITS	4SC2_16S	4SC2_ITS	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	4MR1_16S	4MR1_ITS	4MR2_16S	4MR2_ITS	4SC1_16S	4SC1_ITS	4SC2_16S	4SC2_ITS	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	4MR1_16S	4MR1_ITS	4MR2_16S	4MR2_ITS	4SC1_16S	4SC1_ITS	4SC2_16S	4SC2_ITS	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	4MR1_16S	4MR1_ITS	4MR2_16S	4MR2_ITS	4SC1_16S	4SC1_ITS	4SC2_16S	4SC2_ITS	2 pM Std	2 pM Std	2 pM Std
G	4MR1_16S	4MR1_ITS	4MR2_16S	4MR2_ITS	4SC1_16S	4SC1_ITS	4SC2_16S	4SC2_ITS	20 pM Std	20 pM Std	20 pM Std
H	4MR1_16S	4MR1_ITS	4MR2_16S	4MR2_ITS	4SC1_16S	4SC1_ITS	4SC2_16S	4SC2_ITS

Results:

Average results for the following plates (column 3):

4MR1_16S: 92.23 nanomoles

4MR1_ITS: 53.47 nanomoles

4MR2_16S: 105.34 nanomoles

4MR2_ITS: 66.71 nanomoles

4SC1_16S: 46.64 nanomoles

4SC1_ITS: 47.77 nanomoles

4SC2_16S: 65.97 nanomoles

4SC2_ITS: 55.69 nanomoles

Quantities look good for sequencing. The full result report can be viewed below:

MasterMix (make for 24 plates in 50mL conical tube)

ul/rxn	Reagent	# of rxns	ul needed
3	5X Kapa HiFi Buffer	2500	7500
0.45	10M dNTPs	2500	1125
0.3	Kapa HiFi HotStart DNA Pol	2500	750
7.25	HPLC H2O	2500	18125
11	Total Volume	2500	27500

Add 11 ul to each well of a hard shell, full skirt plate. Seal with bubble strips and store in refrigerator until needed label “NovaSeq4 PCR”. (If doing manually, one needs 2 ul of template and 2 ul of the primers).

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 NovaSeq4_Plate4

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	10	11	12
A	4LVD1_16S	4LVD1_ITS	4LVD2_16S	4LVD2_ITS	4LVD3_16S	4LVD3_ITS	NTC	NTC	NTC
B	4LVD1_16S	4LVD1_ITS	4LVD2_16S	4LVD2_ITS	4LVD3_16S	4LVD3_ITS	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	4LVD1_16S	4LVD1_ITS	4LVD2_16S	4LVD2_ITS	4LVD3_16S	4LVD3_ITS	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	4LVD1_16S	4LVD1_ITS	4LVD2_16S	4LVD2_ITS	4LVD3_16S	4LVD3_ITS	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	4LVD1_16S	4LVD1_ITS	4LVD2_16S	4LVD2_ITS	4LVD3_16S	4LVD3_ITS	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	4LVD1_16S	4LVD1_ITS	4LVD2_16S	4LVD2_ITS	4LVD3_16S	4LVD3_ITS	2 pM Std	2 pM Std	2 pM Std
G	4LVD1_16S	4LVD1_ITS	4LVD2_16S	4LVD2_ITS	4LVD3_16S	4LVD3_ITS	20 pM Std	20 pM Std	20 pM Std
H	4LVD1_16S	4LVD1_ITS	4LVD2_16S	4LVD2_ITS	4LVD3_16S	4LVD3_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)	80	800
2 ul	Primer Premix (10X)	80	160
4 ul	Ultra Pure Water	80	320
16 ul	Total Volume	80	1280

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

	1	2	3	4	5	6	10	11	12
A	4LVD1_16S	4LVD1_ITS	4LVD2_16S	4LVD2_ITS	4LVD3_16S	4LVD3_ITS	NTC	NTC	NTC
B	4LVD1_16S	4LVD1_ITS	4LVD2_16S	4LVD2_ITS	4LVD3_16S	4LVD3_ITS	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	4LVD1_16S	4LVD1_ITS	4LVD2_16S	4LVD2_ITS	4LVD3_16S	4LVD3_ITS	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	4LVD1_16S	4LVD1_ITS	4LVD2_16S	4LVD2_ITS	4LVD3_16S	4LVD3_ITS	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	4LVD1_16S	4LVD1_ITS	4LVD2_16S	4LVD2_ITS	4LVD3_16S	4LVD3_ITS	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	4LVD1_16S	4LVD1_ITS	4LVD2_16S	4LVD2_ITS	4LVD3_16S	4LVD3_ITS	2 pM Std	2 pM Std	2 pM Std
G	4LVD1_16S	4LVD1_ITS	4LVD2_16S	4LVD2_ITS	4LVD3_16S	4LVD3_ITS	20 pM Std	20 pM Std	20 pM Std
H	4LVD1_16S	4LVD1_ITS	4LVD2_16S	4LVD2_ITS	4LVD3_16S	4LVD3_ITS

Results:

Average results for the following plates (column 3):

4LVD1_16S: 112.54 nanomoles

4LVD1_ITS: 24.71 nanomoles

4LVD2_16S: 95.57 nanomoles

4LVD2_ITS: 34.28 nanomoles

4LVD3_16S: 112.47 nanomoles

4LVD3_ITS: 101.03 nanomoles

Quantities look good for sequencing. The full result report can be viewed below:

qPCR NovaSeq4_Plate5

Make 1:1000 dilutions of column 3 from the PCR plates by adding 1 ul to 999 ul TE in a deep well plate:
Use other half of NS4_Plate 4 dilution plate to do NS4_Plate5 dilution plate.

	1	2	3	4	5	6	7	8	9	10	11	12
A	______	______	______	______	______	______	4FB1_16S	4FB1_ITS	4FB2_16S	4FB2_ITS	4FB3_16S	4FB3_ITS
B	______	______	______	______	______	______	4FB1_16S	4FB1_ITS	4FB2_16S	4FB2_ITS	4FB3_16S	4FB3_ITS
C	______	______	______	______	______	______	4FB1_16S	4FB1_ITS	4FB2_16S	4FB2_ITS	4FB3_16S	4FB3_ITS
D	______	______	______	______	______	______	4FB1_16S	4FB1_ITS	4FB2_16S	4FB2_ITS	4FB3_16S	4FB3_ITS
E	______	______	______	______	______	______	4FB1_16S	4FB1_ITS	4FB2_16S	4FB2_ITS	4FB3_16S	4FB3_ITS
F	______	______	______	______	______	______	4FB1_16S	4FB1_ITS	4FB2_16S	4FB2_ITS	4FB3_16S	4FB3_ITS
G	______	______	______	______	______	______	4FB1_16S	4FB1_ITS	4FB2_16S	4FB2_ITS	4FB3_16S	4FB3_ITS
H	______	______	______	______	______	______	4FB1_16S	4FB1_ITS	4FB2_16S	4FB2_ITS	4FB3_16S	4FB3_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)	80	800
2 ul	Primer Premix (10X)	80	160
4 ul	Ultra Pure Water	80	320
16 ul	Total Volume	80	1280

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

	1	2	3	4	5	6	10	11	12
A	4FB1_16S	4FB1_ITS	4FB2_16S	4FB2_ITS	4FB3_16S	4FB3_ITS	NTC	NTC	NTC
B	4FB1_16S	4FB1_ITS	4FB2_16S	4FB2_ITS	4FB3_16S	4FB3_ITS	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	4FB1_16S	4FB1_ITS	4FB2_16S	4FB2_ITS	4FB3_16S	4FB3_ITS	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	4FB1_16S	4FB1_ITS	4FB2_16S	4FB2_ITS	4FB3_16S	4FB3_ITS	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	4FB1_16S	4FB1_ITS	4FB2_16S	4FB2_ITS	4FB3_16S	4FB3_ITS	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	4FB1_16S	4FB1_ITS	4FB2_16S	4FB2_ITS	4FB3_16S	4FB3_ITS	2 pM Std	2 pM Std	2 pM Std
G	4FB1_16S	4FB1_ITS	4FB2_16S	4FB2_ITS	4FB3_16S	4FB3_ITS	20 pM Std	20 pM Std	20 pM Std
H	4FB1_16S	4FB1_ITS	4FB2_16S	4FB2_ITS	4FB3_16S	4FB3_ITS

Results:

Average results for the following plates (column 3):

4FB1_16S: 28.57 nanomoles

4FB1_ITS: 45.42 nanomoles

4FB2_16S: 70.82 nanomoles

4FB2_ITS: 71.52 nanomoles

4FB3_16S: 73.04 nanomoles

4FB3_ITS: 72.32 nanomoles

Quantities look good for sequencing. The full result report can be viewed below: