NovaSeq4 1Step PCR and qPCR

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

4AH1	4AH2
4PA1	4PA2	4PA3	4PA4	4PA5
4MR1	4MR2

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 “PCR1Step”. (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_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)	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	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:

4AH1_ITS:

4AH2_16S:

4AH2_ITS:

4PA1_16S:

4PA1_ITS:

4PA2_16S:

4PA2_ITS:

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 “PCR1Step”. (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	9	10	11	12
A	4PA3_16S	4PA3_ITS	4PA4_16s	4PA4_ITS	4PA5_16S	4PA5_ITS	4MR1_16S	4MR1_ITS	4MR2_16S (Plate3)	4MR2_ITS (Plate3)		NTC
B	4PA3_16S	4PA3_ITS	4PA4_16s	4PA4_ITS	4PA5_16S	4PA5_ITS	4MR1_16S	4MR1_ITS	4MR2_16S (Plate3)	4MR2_ITS (Plate3)	04.0002 pM Std	0.0002 pM Std
C	4PA3_16S	4PA3_ITS	4PA4_16s	4PA4_ITS	4PA5_16S	4PA5_ITS	4MR1_16S	4MR1_ITS	4MR2_16S (Plate3)	4MR2_ITS (Plate3)	0.002 pM Std	0.002 pM Std
D	4PA3_16S	4PA3_ITS	4PA4_16s	4PA4_ITS	4PA5_16S	4PA5_ITS	4MR1_16S	4MR1_ITS	4MR2_16S (Plate3)	4MR2_ITS (Plate3)	0.02 pM Std	0.02 pM Std
E	4PA3_16S	4PA3_ITS	4PA4_16s	4PA4_ITS	4PA5_16S	4PA5_ITS	4MR1_16S	4MR1_ITS	4MR2_16S (Plate3)	4MR2_ITS (Plate3)	0.2 pM Std	0.2 pM Std
F	4PA3_16S	4PA3_ITS	4PA4_16s	4PA4_ITS	4PA5_16S	4PA5_ITS	4MR1_16S	4MR1_ITS	4MR2_16S (Plate3)	4MR2_ITS (Plate3)	2 pM Std	2 pM Std
G	4PA3_16S	4PA3_ITS	4PA4_16s	4PA4_ITS	4PA5_16S	4PA5_ITS	4MR1_16S	4MR1_ITS	4MR2_16S (Plate3)	4MR2_ITS (Plate3)	20 pM Std	20 pM Std
H	4PA3_16S	4PA3_ITS	4PA4_16s	4PA4_ITS	4PA5_16S	4PA5_ITS	4MR1_16S	4MR1_ITS	4MR2_16S (Plate3)	4MR2_ITS (Plate3)

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	4MR1_16S	4MR1_ITS	NTC	NTC	NTC
B	4PA3_16S	4PA3_ITS	4PA4_16s	4PA4_ITS	4PA5_16S	4PA5_ITS	4MR1_16S	4MR1_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	4MR1_16S	4MR1_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	4MR1_16S	4MR1_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	4MR1_16S	4MR1_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	4MR1_16S	4MR1_ITS	2 pM Std	2 pM Std	2 pM Std
G	4PA3_16S	4PA3_ITS	4PA4_16s	4PA4_ITS	4PA5_16S	4PA5_ITS	4MR1_16S	4MR1_ITS	20 pM Std	20 pM Std	20 pM Std
H	4PA3_16S	4PA3_ITS	4PA4_16s	4PA4_ITS	4PA5_16S	4PA5_ITS	4MR1_16S	4MR1_ITS

Results:

Average results for the following plates (column 3):

4PA3_16S:

4PA3_ITS:

4PA4_16S:

4PA4_ITS:

4PA5_16S:

4PA5_ITS:

4MR1_16S:

4MR2_ITS: