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New Control Samples:

Start Reaction Plate Setup:

Add Ultrapure H2O to the reaction plates in the following pattern:

NOTE: Water in the master mix has been adjusted to a lower amount. The plate set up below will replace the water usually added to the master mix while also showing if a decrease in water and an increase in template will increase PCR yields.

	1	2
A	6ul	4ul
B	6ul	4ul
C	6ul	4ul
D	6ul	4ul
E	6ul	4ul
F	6ul	4ul
G	6ul	4ul
H	6ul	4ul

MasterMix

ul/rxn	Reagent	# of rxns	ul needed
3	5X Kapa HiFi Buffer	24	120
0.45	10M dNTPs	24	18
0.3	Kapa HiFi HotStart DNA Pol	24	12
0.25	HPLC H2O	24	10
1	ISD	24	40
5	Total Volume	24	200

Add 5 ul to each well of a hard shell, full skirt plate.
Add 10 ng/ul Mock Community to the plate in the following pattern:

	1	2	3
A	2ul	4ul	8ul
B	2ul	4ul	8ul
C	2ul	4ul	8ul
D	2ul	4ul	8ul
E	2ul	4ul	8ul
F	2ul	4ul	8ul
G	2ul	4ul	8ul
H	2ul	4ul	8ul

Add 2 ul 1-step primers (from columns 7-9): 16S0A8
Seal with bubble seals. Vortex briefly. Spin down.
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 (Do not reuse any tips):

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:

Make 3 separate 1:1000 dilutions 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	RA6	RA14	RA22	LL1A_J	WG3A_S	LY2A_S	MCA_2	MCA_4	MCA_8	NTC	NTC	NTC
B	RA7	RA15	RA23	LL1B_J	WG3B_S	LY2B_S	MCB_2	MCB_4	MCB_8	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	RA8	RA16	RA24	LL2A_J	WG4A_S	LY3B_S	MCC_2	MCC_4	MCC_8	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	RA9	RA17	RA25	LL2B_J	WG4B_S	LY1A_S	MCD_2	MCD_4	MCD_8	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	RA10	RA18	RA26	LL4A_J	WG2A_S	LY1B_S	MCA_2_clean	MCA_4_clean	MCA_8_clean	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	RA11	RA19	RA27	LL4B_J	WG2B_S	LY4A_S	MCB_2_clean	MCB_4_clean	MCB_8_clean	2 pM Std	2 pM Std	2 pM Std
G	RA12	RA20	RA28	LL3A_J	WG1A_S	LY4B_S	MCC_2_clean	MCC_4_clean	MCC_8_clean
H	RA13	RA21	RA29	LL3B_J	WG1B_S	BLANK	MCD_2_clean	MCD_4_clean	MCD_8_clean
Plate	5LVD5	5LVD5	5LVD5	5DG5	5DG5	5DG5	Controls	Controls	Controls
Column	1	2	3	4	5	6

Add 16 ul of Illumina Library Quantification MasterMix to each well of 4 plates:

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	RA6	RA14	RA22	RA6	RA14	RA22	RA6	RA14	RA22	NTC	NTC	NTC
B	RA7	RA15	RA23	RA7	RA15	RA23	RA7	RA15	RA23	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	RA8	RA16	RA24	RA8	RA16	RA24	RA8	RA16	RA24	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	RA9	RA17	RA25	RA9	RA17	RA25	RA9	RA17	RA25	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	RA10	RA18	RA26	RA10	RA18	RA26	RA10	RA18	RA26	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	RA11	RA19	RA27	RA11	RA19	RA27	RA11	RA19	RA27	2 pM Std	2 pM Std	2 pM Std
G	RA12	RA20	RA28	RA12	RA20	RA28	RA12	RA20	RA28	20 pM Std	20 pM Std	20 pM Std
H	RA13	RA21	RA29	RA13	RA21	RA29	RA13	RA21	RA29

	1	2	3	4	5	6	7	8	9	10	11	12
A	LL1A_J	WG3A_S	LY2A_S	LL1A_J	WG3A_S	LY2A_S	LL1A_J	WG3A_S	LY2A_S	NTC	NTC	NTC
B	LL1B_J	WG3B_S	LY2B_S	LL1B_J	WG3B_S	LY2B_S	LL1B_J	WG3B_S	LY2B_S	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	LL2A_J	WG4A_S	LY3B_S	LL2A_J	WG4A_S	LY3B_S	LL2A_J	WG4A_S	LY3B_S	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	LL2B_J	WG4B_S	LY1A_S	LL2B_J	WG4B_S	LY1A_S	LL2B_J	WG4B_S	LY1A_S	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	LL4A_J	WG2A_S	LY1B_S	LL4A_J	WG2A_S	LY1B_S	LL4A_J	WG2A_S	LY1B_S	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	LL4B_J	WG2B_S	LY4A_S	LL4B_J	WG2B_S	LY4A_S	LL4B_J	WG2B_S	LY4A_S	2 pM Std	2 pM Std	2 pM Std
G	LL3A_J	WG1A_S	LY4B_S	LL3A_J	WG1A_S	LY4B_S	LL3A_J	WG1A_S	LY4B_S	20 pM Std	20 pM Std	20 pM Std
H	LL3B_J	WG1B_S	BLANK	LL3B_J	WG1B_S	BLANK	LL3B_J	WG1B_S	BLANK

	1	2	3	4	5	6	7	8	9	10	11	12
A	MCA_2	MCA_4	MCA_8	MCA_2	MCA_4	MCA_8	MCA_2	MCA_4	MCA_8	NTC	NTC	NTC
B	MCB_2	MCB_4	MCB_8	MCB_2	MCB_4	MCB_8	MCB_2	MCB_4	MCB_8	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	MCC_2	MCC_4	MCC_8	MCC_2	MCC_4	MCC_8	MCC_2	MCC_4	MCC_8	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	MCD_2	MCD_4	MCD_8	MCD_2	MCD_4	MCD_8	MCD_2	MCD_4	MCD_8	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	MCA_2_clean	MCA_4_clean	MCA_8_clean	MCA_2_clean	MCA_4_clean	MCA_8_clean	MCA_2_clean	MCA_4_clean	MCA_8_clean	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	MCB_2_clean	MCB_4_clean	MCB_8_clean	MCB_2_clean	MCB_4_clean	MCB_8_clean	MCB_2_clean	MCB_4_clean	MCB_8_clean	2 pM Std	2 pM Std	2 pM Std
G	MCC_2_clean	MCC_4_clean	MCC_8_clean	MCC_2_clean	MCC_4_clean	MCC_8_clean	MCC_2_clean	MCC_4_clean	MCC_8_clean	20 pM Std	20 pM Std	20 pM Std
H	MCD_2_clean	MCD_4_clean	MCD_8_clean	MCD_2_clean	MCD_4_clean	MCD_8_clean	MCD_2_clean	MCD_4_clean	MCD_8_clean
	Controls	Controls	Controls	Controls	Controls	Controls	Controls	Controls	Controls

Results:

5LVD5 Results:

5DG5 Results:

MC_MagBead Results:

Pooling and Sequencing:

Pool 2 ul from all original product plates. Make 3 1:1000 dilutions of pool and qPCR as usual. Dilute pool to 1nM. Dilute to loading concentration with 20% PhiX.

qPCR Master Mix:

Add 16 ul of Illumina Library Quantification MasterMix to each well of 4 plates:

ul/rxn	Reagent	# of rxns	ul needed
10 ul	KAPA SYBR FAST qPCR MM (2X)	40	800
2 ul	Primer Premix (10X)	40	160
4 ul	Ultra Pure Water	40	320
16 ul	Total Volume	40	1280

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

	1	2	10	11	12
A	LRIII_MC_MB_Pool1	LRIII_MC_MB_Pool3	NTC	NTC	NTC
B	LRIII_MC_MB_Pool1		0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	LRIII_MC_MB_Pool1		0.002 pM Std	0.002 pM Std	0.002 pM Std
D	LRIII_MC_MB_Pool2		0.02 pM Std	0.02 pM Std	0.02 pM Std
E	LRIII_MC_MB_Pool2		0.2 pM Std	0.2 pM Std	0.2 pM Std
F	LRIII_MC_MB_Pool2		2 pM Std	2 pM Std	2 pM Std
G	LRIII_MC_MB_Pool3		20 pM Std	20 pM Std	20 pM Std
H	LRIII_MC_MB_Pool3

Results:

iSeq Run:

Dilute to 1 nM based off qPCR results. qPCR results are in pM, but 1:1000 dilution used. The results are effectively in nM for pool.

1000/Results = ul of Pool to Add

1000/73 = 13.7uL of Pool to Add

1000 - uL of Pool to Add = ul of “10 mM Tris 8.5” to Add

1000- 13.7 = 986.3 uL of 10mM Tris 8.5

Dilute to loading concentration with 20% PhiX.

Loading pool is:

78ul 10mM Tris HCL pH 8, 16 ul 50 pM PhiX, 6 ul from above pool.

	1	2	3
A	2ul	4ul	8ul
B	2ul	4ul	8ul
C	2ul	4ul	8ul
D	2ul	4ul	8ul
E	2ul	4ul	8ul
F	2ul	4ul	8ul
G	2ul	4ul	8ul
H	2ul	4ul	8ul

	1	2	3
A	2ul	4ul	8ul
B	2ul	4ul	8ul
C	2ul	4ul	8ul
D	2ul	4ul	8ul
E	2ul	4ul	8ul
F	2ul	4ul	8ul
G	2ul	4ul	8ul
H	2ul	4ul	8ul

	1	2	3
A	2ul	4ul	8ul
B	2ul	4ul	8ul
C	2ul	4ul	8ul
D	2ul	4ul	8ul
E	2ul	4ul	8ul
F	2ul	4ul	8ul
G	2ul	4ul	8ul
H	2ul	4ul	8ul