Page Comparison

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4AH1	4AH2
4PA1	4PA2	4PA3	4PA4	4PA5	4PA6
4MR1	4MR2
4SC1	4SC2
4LVD1	4LVD2	4LVD3

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

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	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)	80100	8001000
2 ul	Primer Premix (10X)	80100	160200
4 ul	Ultra Pure Water	80100	320400
16 ul	Total Volume	80100	12801600

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

	1	2	3	4	5	6	7	8	9	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		20 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:

4MR2_ITS:

4SC1_16S:

4SC1_ITS:

4SC2_16S:

4SC2_ITS:

MasterMix (make for 10 plates in 50mL conical tube)

ul/rxn	Reagent	# of rxns	ul needed
3	5X Kapa HiFi Buffer	1000	3000
0.45	10M dNTPs	1000	450
0.3	Kapa HiFi HotStart DNA Pol	1000	300
7.25	HPLC H2O	1000	7250
11	Total Volume	1000	11000

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_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	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:

4LVD1_ITS:

4LVD2_16S:

4LVD2_ITS:

4LVD3_16S:

4LVD3_ITS:

Versions Compared

Old Version 8

New Version 9

Key

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

Results:

MasterMix (make for 10 plates in 50mL conical tube)

MagBead Cleanup:

qPCR NovaSeq4_Plate1

Results: