Page Comparison

NS5 Plates

...

5AL1

...

5AL2

...

5AL3

...

5AL4

...

5AL5

...

5AL6

...

5AL7

...

5AL8

...

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

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5AL1

...

16S0A1

...

ITS0A1

...

16S0B1

...

ITS0B1

...

5AL2

...

16S0C1

...

ITS0C1

...

16S0D1

...

ITS0D1

...

5AL3

...

16S0E1

...

ITS0E1

...

16S0F1

...

ITS0F1

...

5AL4

...

16S0G1

...

ITS0G1

...

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)

...

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:

View file

name	NS5_SET1.csv

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:

View file

name	NS5_SET2.csv

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	10	11

12

A

4AH1_16S

4AH1_ITS

4AH2_16S

4AH2_ITS

4PA1_16S

4PA1_ITS

4PA2_16S

4PA2_ITS

NTC

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

4AH1

5LVD3_16S_Col3

4AH1

5LVD4_16S_

ITS

Col3

4AH2

5LVD5_16S_Col3

4AH2

5LVD1_ITS_Col3

4PA1

5LVD2_ITS_

16S

Col3

4PA1

5LVD3_ITS_Col3

4PA2

5LVD4_ITS_

16S

Col3

4PA2

5LVD5_ITS_Col3

5FB1_16S_Col3

0

C

4AH1_16S

4AH1_ITS

4AH2_16S

4AH2_ITS

4PA1_16S

4PA1_ITS

4PA2_16S

4PA2_ITS

0.002 pM Std

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

D

4AH1

5LVD3_16S

4AH1

_

ITS

Col3

4AH2

5LVD4_16S

4AH2

_

ITS

Col3

4PA1

5LVD5_16S_Col3

4PA1

5LVD1_ITS

4PA2

_

16S

Col3

4PA2

5LVD2_ITS

0.02 pM Std

E

4AH1_16S

4AH1_ITS

4AH2_16S

4AH2_ITS

4PA1_16S

4PA1_ITS

4PA2_16S

4PA2_ITS

_Col3

5LVD3_ITS_Col3

5LVD4_ITS_Col3

5LVD5_ITS_Col3

5FB1_16S_Col3

0.

2

02 pM Std

0.

2

F

4AH1

02 pM Std

0.2 pM Std

E

5LVD3_16S

4AH1

_

ITS

Col3

4AH2

5LVD4_16S

4AH2

_

ITS

Col3

4PA1

5LVD5_16S_Col3

4PA1

5LVD1_ITS

4PA2

_

16S

Col3

4PA2

5LVD2_ITS

2 pM Std

G

4AH1_16S

4AH1_ITS

4AH2_16S

4AH2_ITS

4PA1_16S

4PA1_ITS

4PA2_16S

4PA2_ITS

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

...

9

...

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:

View file

name	NovaSeq4_Plate1.csv

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)

...

_Col3

5LVD3_ITS_Col3

5LVD4_ITS_Col3

5LVD5_ITS_Col3

5FB1_16S_Col3

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

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

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:

View file

name	NS5_SET3.csv

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	10	11	12

A

4PA3_16S

4PA3_ITS

4PA4_16s

4PA4_ITS

4PA5_16S

4PA5_ITS

4PA6_16S

4PA6_ITS


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

4PA3

5FB2_16S_Col3

5FB3_16S_Col3

4PA3

5FB4_16S_

ITS

Col3

4PA4

5FB5_16S_

16s

Col3

4PA4

5FB1_ITS_Col3

4PA5

5FB2_ITS_

16S

Col3

4PA5

5FB3_ITS_Col3

4PA6

5FB4_ITS_

16S

Col3

4PA6

5FB5_ITS

_Col3

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

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

D

4PA3

5FB2_16S_Col3

5FB3_16S_Col3

4PA3

5FB4_16S_

ITS

Col3

4PA4

5FB5_16S_

16s

Col3

4PA4

5FB1_ITS_Col3

4PA5

5FB2_ITS_

16S

Col3

4PA5

5FB3_ITS_Col3

4PA6

5FB4_ITS_

16S

Col3

4PA6

5FB5_ITS

_Col3

0.02 pM Std

E

4PA3_16S

4PA3_ITS

4PA4_16s

4PA4_ITS

4PA5_16S

4PA5_ITS

4PA6_16S

4PA6_ITS

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

F

4PA3

5FB2_16S_Col3

5FB3_16S_Col3

4PA3

5FB4_16S_

ITS

Col3

4PA4

5FB5_16S_

16s

Col3

4PA4

5FB1_ITS_Col3

4PA5

5FB2_ITS_

16S

Col3

4PA5

5FB3_ITS_Col3

4PA6

5FB4_ITS_

16S

Col3

4PA6

5FB5_ITS_Col3

G

4PA3_16S

4PA3_ITS

4PA4_16s

4PA4_ITS

4PA5_16S

4PA5_ITS

4PA6_16S

4PA6_ITS

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

H

4PA3

5FB2_16S_Col3

5FB3_16S_Col3

4PA3

5FB4_16S_

ITS

Col3

4PA4

5FB5_16S_

16s

Col3

4PA4

5FB1_ITS_Col3

4PA5

5FB2_ITS_

16S

Col3

4PA5

5FB3_ITS_Col3

4PA6

5FB4_ITS_

16S

Col3

4PA6

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)

100

110

1000

1100
2 ul	Primer Premix (10X)

100

110

200

220
4 ul	Ultra Pure Water

100

110

400

440
16 ul	Total Volume

100

110

1600

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

4PA3

5FB2_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

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

D

4PA3_16S

4PA3_ITS

4PA4_16s

4PA4_ITS

4PA5_16S

4PA5_ITS

4PA6_16S

4PA6_ITS

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

F

4PA3_16S

4PA3_ITS

4PA4_16s

4PA4_ITS

4PA5_16S

4PA5_ITS

4PA6_16S

4PA6_ITS

2 pM Std

G

4PA3_16S

4PA3_ITS

4PA4_16s

4PA4_ITS

4PA5_16S

4PA5_ITS

4PA6_16S

4PA6_ITS

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:

View file

name	NovaSeq4_Plate2.csv

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)

...

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

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

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

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

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

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

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:

View file

name	NS5_SET4.csv

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	10	11	12
A

4MR1

5DG1_16S_Col3

4MR1

5DG2_16S_

ITS

Col3

4MR2

5DG3_16S

4MR2_ITS

4SC1_16S

4SC1_ITS

4SC2_16S

4SC2_ITS

_Col3	5DG4_16S_Col3	5DG5_16S_Col3	5DG1_ITS_Col3	5DG2_ITS_Col3	5DG3_ITS_Col3	5DG4_ITS_Col3			NTC
B

4MR1

5DG1_16S_Col3

4MR1

5DG2_16S_

ITS

Col3

4MR2

5DG3_16S

4MR2_ITS

4SC1_16S

4SC1_ITS

4SC2_16S

4SC2_ITS

_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

4MR1

5DG1_16S_Col3

4MR1

5DG2_16S_

ITS

Col3

4MR2

5DG3_16S

4MR2_ITS

4SC1_16S

4SC1_ITS

4SC2_16S

4SC2_ITS

_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

4MR1

5DG1_16S_Col3

4MR1

5DG2_16S_

ITS

Col3

4MR2

5DG3_16S

4MR2_ITS

4SC1_16S

4SC1_ITS

4SC2_16S

4SC2_ITS

_Col3

5DG4_16S_Col3

5DG5_16S_Col3

5DG1_ITS_Col3

5DG2_ITS_Col3

5DG3_ITS_Col3

5DG4_ITS_Col3

0.02 pM Std

E

4MR1


E	5DG1_16S_Col3	5DG2_16S_Col3

4MR1

5DG3_16S_

ITS

Col3

4MR2

5DG4_16S

4MR2

_Col3

5DG5_16S_Col3

5DG1_ITS

4SC1_16S

4SC1_ITS

4SC2_16S

4SC2_ITS

_Col3	5DG2_ITS_Col3	5DG3_ITS_Col3	5DG4_ITS_Col3		0.2 pM Std	0.2 pM Std
F

4MR1

5DG1_16S_Col3

4MR1

5DG2_16S_

ITS

Col3

4MR2

5DG3_16S

4MR2_ITS

4SC1_16S

4SC1_ITS

4SC2_16S

4SC2_ITS

_Col3

5DG4_16S_Col3

5DG5_16S_Col3

5DG1_ITS_Col3

5DG2_ITS_Col3

5DG3_ITS_Col3

5DG4_ITS_Col3

2 pM Std

G

4MR1


G	5DG1_16S_Col3	5DG2_16S_Col3

4MR1

5DG3_16S_

ITS

Col3

4MR2

5DG4_16S

4MR2

_Col3

5DG5_16S_Col3

5DG1_ITS

4SC1_16S

4SC1_ITS

4SC2_16S

4SC2_ITS

_Col3	5DG2_ITS_Col3	5DG3_ITS_Col3	5DG4_ITS_Col3		20 pM Std	20 pM Std
H

4MR1

5DG1_16S_Col3

4MR1

5DG2_16S_

ITS

Col3

4MR2

5DG3_16S

4MR2_ITS

4SC1_16S

4SC1_ITS

4SC2_16S

4SC2_ITS

_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)

100

110

1000

1100
2 ul	Primer Premix (10X)

100

110

200

220
4 ul	Ultra Pure Water

100

110

400

440
16 ul	Total Volume

100

110

1600

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

...

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:

View file

name	NovaSeq4_Plate3.csv

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)

...

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	42
5DG2_16S_Col3	31.72
5DG3_16S_Col3	76.88
5DG4_16S_Col3	55.24
5DG5_16S_Col3	48.91
5DG1_ITS_Col3	37.74
5DG2_ITS_Col3	37.41
5DG3_ITS_Col3	34.67
5DG4_ITS_Col3	17.29

Full qPCR results below:

View file

name	NS5_SET5.csv

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	10	11	12
A

4LVD1

5DG5_ITS_Col3

5RD1_16S_Col1

5RD1_16S_Col2

5RD1_16S_Col3

4LVD1

5RD1_16S_

ITS

Col4

4LVD2

5RD1_16S_Col5

4LVD2

5RD1_16S_

ITS

Col6

4LVD3

5RD1_16S_Col7

4LVD30

5RD1_ITS_Col1

NTC

B

4LVD1_16S

4LVD1_ITS

4LVD2_16S

4LVD2_ITS

4LVD3_16S

4LVD3_ITS

C

4LVD1_16S

4LVD1_ITS

4LVD2_16S

4LVD2_ITS

4LVD3_16S

4LVD3_ITS

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

D

4LVD1_16S

4LVD1_ITS

4LVD2_16S

4LVD2_ITS

4LVD3_16S

4LVD3_ITS

E

4LVD1_16S

4LVD1_ITS

4LVD2_16S

4LVD2_ITS

4LVD3_16S

4LVD3_ITS

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

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

F

4LVD1_16S

4LVD1_ITS

4LVD2_16S

4LVD2_ITS

4LVD3_16S

4LVD3_ITS

G

4LVD1_16S

4LVD1_ITS

4LVD2_16S

4LVD2_ITS

4LVD3_16S

4LVD3_ITS

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

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

H

4LVD1_16S

4LVD1_ITS

4LVD2_16S

4LVD2_ITS

4LVD3_16S

4LVD3_ITS

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)

80

110

800

1100
2 ul	Primer Premix (10X)

80

110

160

220
4 ul	Ultra Pure Water

80

110

320

440
16 ul	Total Volume

80

110

1280

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

4LVD1_16S

4LVD1_ITS

4LVD2_16S

4LVD2_ITS

4LVD3_16S

4LVD3_ITS

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

4LVD1

5DG5_ITS_Col3

5RD1_16S_Col1

4LVD1

5RD1_16S_

ITS

Col2

4LVD2

5RD1_16S_Col3

4LVD2

5RD1_16S_

ITS

Col4

4LVD3

5RD1_16S_Col5

4LVD3

5RD1_16S_

ITS

Col6	5RD1_16S_Col7	5RD1_ITS_Col1	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

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

D

4LVD1

5DG5_ITS_Col3

5RD1_16S_Col1

4LVD1

5RD1_16S_

ITS

Col2

4LVD2

5RD1_16S_Col3

4LVD2

5RD1_16S_

ITS

Col4

4LVD3

5RD1_16S_Col5

4LVD3

5RD1_16S_

ITS

Col6	5RD1_16S_Col7	5RD1_ITS_Col1	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

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

F

4LVD1

5DG5_ITS_Col3

5RD1_16S_Col1

4LVD1

5RD1_16S_

ITS

Col2

4LVD2

5RD1_16S_Col3

4LVD2

5RD1_16S_

ITS

Col4

4LVD3

5RD1_16S_Col5

4LVD3

5RD1_16S_

ITS

Col6	5RD1_16S_Col7	5RD1_ITS_Col1	2 pM Std	2 pM Std	2 pM Std
G

4LVD1_16S

4LVD1_ITS

4LVD2_16S

4LVD2_ITS

4LVD3_16S

4LVD3_ITS

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

H

4LVD1

5DG5_ITS_Col3

5RD1_16S_Col1

5RD1_16S_Col2

5RD1_16S_Col3

4LVD1

5RD1_16S_

ITS

Col4

4LVD2

5RD1_16S_Col5

4LVD2

5RD1_16S_

ITS

Col6

4LVD3

5RD1_16S_Col7

4LVD3

5RD1_ITS_Col1

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

...

Results:

Plate Name	Average Result (nanomoles)
5DG5_ITS_Col3	27.22
5RD1_16S_Col1	2.79
5RD1_16S_Col2	0.573
5RD1_16S_Col3	2.64
5RD1_16S_Col4	8.07
5RD1_16S_Col5	10.31
5RD1_16S_Col6	4.6
5RD1_16S_Col7	13.52
5RD1_ITS_Col1	5.66

Full qPCR results below:

View file

name	NovaSeq4NS5_Plate4SET6.csv

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:
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

...

7

...

8

...

9

...

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:

...

	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	3.89
5RD1_ITS_Col3	5.49
5RD1_ITS_Col4	5.19
5RD1_ITS_Col5	9.37
5RD1_ITS_Col6	8.81
5RD1_ITS_Col7	16.43
Ha_Yoon_16S_ITS_Col1	19.16
Ha_Yoon_16S_ITS_Col6	18.35
Ha_Yoon_16S_ITS_Col12	15.73

Full qPCR results below:

View file

name	NS5_SET7.csv

Page Comparison

Versions Compared

Old Version 1

New Version Current

Key

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

MagBead Cleanup:

NS5 Plates

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

MagBead Cleanup:

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

MagBead Cleanup:

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

MagBead Cleanup:

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

MagBead Cleanup:

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

MagBead Cleanup:

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

MagBead Cleanup:

qPCR NovaSeq5_Set1

Results:

qPCR NovaSeq5_Set2

Results:

qPCR NovaSeq5_Set3

Results:

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

MagBead Cleanup:

Results:

qPCR NovaSeq5_Set4

Results:

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

MagBead Cleanup:

Results:

qPCR NovaSeq5_Set5

Results:

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

MagBead Cleanup:

Results:

qPCR NovaSeq5_Set6

Results:

Results:

qPCR

NovaSeq5_

Set7

Results:

Results: