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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

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)

...

Some plates were cleaned up using the Nimbus platform protocol “AxyPrep MagBead PCR1 No MM”

Manually, it was done:

...

F

...

 

...

2 pM Std

...

2 pM Std

...

2 pM Std

...

G

...

 

...

20 pM Std

...

20 pM Std

...

20 pM Std

...

H

...

 

...

 

...

 

...

 

  • 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

...

 

...

NTC

...

NTC

...

NTC

...

B

...

 

...

0.0002 pM Std

...

0.0002 pM Std

...

0.0002 pM Std

...

C

...

 

...

0.002 pM Std

...

0.002 pM Std

...

0.002 pM Std

...

D

...

 

...

0.02 pM Std

...

0.02 pM Std

...

0.02 pM Std

...

E

...

 

...

0.2 pM Std

...

0.2 pM Std

...

0.2 pM Std

...

F

...

 

...

2 pM Std

...

2 pM Std

...

2 pM Std

...

G

...

 

...

20 pM Std

...

20 pM Std

...

20 pM Std

...

H

...

 

...

 

...

 

...

 

Results:

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

...

  • 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 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

...

 

...

NTC

...

NTC

...

NTC

...

B

...

 

...

0.0002 pM Std

...

0.0002 pM Std

...

0.0002 pM Std

...

C

...

 

...

0.002 pM Std

...

0.002 pM Std

...

0.002 pM Std

...

D

...

 

...

0.02 pM Std

...

0.02 pM Std

...

0.02 pM Std

...

E

...

 

...

0.2 pM Std

...

0.2 pM Std

...

0.2 pM Std

  • 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

16S0B2

ITS0B2

5AL6

16S0C2

ITS0C2

16S0D2

ITS0D2

5AL7

16S0E2

ITS0E2

16S0F2

ITS0F2

5AL8

16S0G2

ITS0G2

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

5AL5

5LVD1

16S0A2

16S0A3

ITS0A2

ITS0A3

16S0B2

16S0B3

ITS0B2

ITS0B3

5AL6

5LVD2

16S0C2

16S0C3

ITS0C2

ITS0C3

16S0D2

16S0D3

ITS0D2

ITS0D3

5AL7

5LVD3

16S0E2

16S0E3

ITS0E2

ITS0E3

16S0F2

16S0F3

ITS0F2

ITS0F3

5AL8

5LVD4

16S0G2

16S0G3

ITS0G2

ITS0G3

16S0H2

16S0H3

ITS0H2

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”

...

  • 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 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

...

10

...

11

...

12

...

A

...

 

...

 

...

 

...

NTC

...

B

...

 

...

 

...

04.0002 pM Std

...

0.0002 pM Std

...

C

...

 

...

 

...

0.002 pM Std

...

0.002 pM Std

...

D

...

 

...

 

...

0.02 pM Std

...

0.02 pM Std

...

E

...

 

...

 

...

0.2 pM Std

...

0.2 pM Std

...

F

...

 

...

 

...

2 pM Std

...

2 pM Std

...

G

...

 

...

 

...

20 pM Std

...

20 pM Std

...

H

...

 

...

 

...

 

...

 

  • 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

...

9

...

10

...

11

...

12

...

A

...

 

...

NTC

...

NTC

...

NTC

...

B

...

 

...

0.0002 pM Std

...

0.0002 pM Std

...

0.0002 pM Std

...

C

...

 

...

0.002 pM Std

...

0.002 pM Std

...

0.002 pM Std

...

D

...

 

...

0.02 pM Std

...

0.02 pM Std

...

0.02 pM Std

...

E

...

 

...

0.2 pM Std

...

0.2 pM Std

...

0.2 pM Std

...

F

...

 

...

2 pM Std

...

2 pM Std

...

2 pM Std

...

G

...

 

...

20 pM Std

...

20 pM Std

...

20 pM Std

...

H

...

 

...

 

...

 

...

 

Results:

...

  • 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

16S0B4

ITS0B4

5FB1

16S0C4

ITS0C4

16S0D4

ITS0D4

5FB2

16S0E4

ITS0E4

16S0F4

ITS0F4

5FB3

16S0G4

ITS0G4

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

5LVD1

5FB4

16S0A3

16S0A5

ITS0A3

ITS0A5

16S0B3

16S0B5

ITS0B3

ITS0B5

5LVD2

5FB5

16S0C3

16S0C5

ITS0C3

ITS0C5

16S0D3

16S0D5

ITS0D3

ITS0D5

5LVD3

5DG1

16S0E3

16S0E5

ITS0E3

ITS0E5

16S0F3

16S0F5

ITS0F3

ITS0F5

5LVD4

5DG2

16S0G3

16S0G5

ITS0G3

ITS0G5

16S0H3

16S0H5

ITS0H3

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”

...

  • 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 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

...

10

...

11

...

12

...

A

...

 

...

 

...

 

...

NTC

...

B

...

 

...

 

...

04.0002 pM Std

...

0.0002 pM Std

...

C

...

 

...

 

...

0.002 pM Std

...

0.002 pM Std

...

D

...

 

...

 

...

0.02 pM Std

...

0.02 pM Std

...

E

...

 

...

 

...

0.2 pM Std

...

0.2 pM Std

...

F

...

 

...

 

...

2 pM Std

...

2 pM Std

...

G

...

 

...

 

...

20 pM Std

...

20 pM Std

...

H

...

 

...

 

...

 

...

 

  • 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

...

9

...

10

...

11

...

12

...

A

...

 

...

NTC

...

NTC

...

NTC

...

B

...

 

...

0.0002 pM Std

...

0.0002 pM Std

...

0.0002 pM Std

...

C

...

 

...

0.002 pM Std

...

0.002 pM Std

...

0.002 pM Std

...

D

...

 

...

0.02 pM Std

...

0.02 pM Std

...

0.02 pM Std

...

E

...

 

...

0.2 pM Std

...

0.2 pM Std

...

0.2 pM Std

...

F

...

 

...

2 pM Std

...

2 pM Std

...

2 pM Std

...

G

...

 

...

20 pM Std

...

20 pM Std

...

20 pM Std

...

H

...

 

...

 

...

 

...

 

Results:

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

...

16S0B4

...

ITS0B4

...

5FB1

...

16S0C4

...

ITS0C4

...

16S0D4

...

ITS0D4

...

5FB2

...

16S0E4

...

ITS0E4

...

16S0F4

...

ITS0F4

...

5FB3

...

16S0G4

...

ITS0G4

...

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)

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 plateStore 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

16S0B6

ITS0B6

5DG4

16S0C6

ITS0C6

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:

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

 

1

2

3

4

5

6

7

8

9

10

11

12

A

 

NTC

NTC

NTC

B

 

0.0002 pM Std

0.0002 pM Std

0.0002 pM Std

C

 

0.002 pM Std

0.002 pM Std

0.002 pM Std

D

 

0.02 pM Std

0.02 pM Std

0.02 pM Std

E

 

0.2 pM Std

0.2 pM Std

0.2 pM Std

F

 

2 pM Std

2 pM Std

2 pM Std

G

 

 

1

2

3

4

5

6

7

8

9

10

11

12

A

 

 

 

NTC

B

 

 

04.0002 pM Std

0.0002 pM Std

C

 

 

0.002 pM Std

0.002 pM Std

D

 

 

0.02 pM Std

0.02 pM Std

E

 

 

0.2 pM Std

0.2 pM Std

F

 

 

2 pM Std

2 pM Std

G

 

 

20 pM Std

20 pM Std

H

 

 

 

 

  • 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

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:

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

...

16S0B5

...

ITS0B5

...

5FB5

...

16S0C5

...

ITS0C5

...

16S0D5

...

ITS0D5

...

5DG1

...

16S0E5

...

ITS0E5

...

16S0F5

...

ITS0F5

...

5DG2

...

16S0G5

...

ITS0G5

...

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)

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

10

11

12

A

 

 

 

NTC

B

 

 

04.0002 pM Std

0.0002 pM Std

C

 

 

0.002 pM Std

0.002 pM Std

D

 

 

0.02 pM Std

0.02 pM Std

E

 

 

0.2 pM Std

0.2 pM Std

F

 

 

2 pM Std

2 pM Std

G

 

 

20 pM Std

20 pM Std

H

 

Plate Name

Average Result (nanomoles)

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

Full qPCR results below:

 

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

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

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)

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:

B

 

0.0002 pM Std

0.0002 pM Std

0.0002 pM Std

C

 

0.002

 

1

2

3

4

5

6

7

8

9

10

11

12

A

 

NTC

NTC

NTC

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.

002

0002 pM Std

0.

002

E

 

0.2

0002 pM Std

D

 

0.02 pM Std

0.02 pM Std

0.02 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.

2

002 pM Std

0.

2

002 pM Std

F

 

2 pM Std

2 pM Std

2 pM Std

G

 

20 pM Std

20 pM Std

20 pM Std

H

 

 

 

 

Results:

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

...

16S0B6

...

ITS0B6

...

5DG4

...

16S0C6

...

ITS0C6

...

16S0D6

...

ITS0D6

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 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

...

10

...

11

...

12

...

A

...

 

...

 

...

 

...

NTC

...

B

...

 

...

 

...

04.0002 pM Std

...

0.0002 pM Std

...

C

...

 

...

 

...

0.002 pM Std

...

0.002 pM Std

...

D

...

 

...

 

...

0.02 pM Std

...

0.02 pM Std

...

E

...

 

...

 

...

0.2 pM Std

...

0.2 pM Std

...

F

...

 

...

 

...

2 pM Std

...

2 pM Std

...

G

...

 

...

 

...

20 pM Std

...

20 pM Std

...

H

...

 

...

 

...

 

...

 

  • 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

...

9

...

10

...

11

...

12

...

A

...

 

...

NTC

...

NTC

...

NTC

...

B

...

 

...

0.0002 pM Std

...

0.0002 pM Std

...

0.0002 pM Std

...

C

...

 

...

0.002 pM Std

...

0.002 pM Std

...

0.002 pM Std

...

D

...

 

...

0.02 pM Std

...

0.02 pM Std

...

0.02 pM Std

...

E

...

 

...

0.2 pM Std

...

0.2 pM Std

...

0.2 pM Std

...

F

...

 

...

2 pM Std

...

2 pM Std

...

2 pM Std

...

G

...

 

...

20 pM Std

...

20 pM Std

...

20 pM Std

...

H

...

 

...

 

...

 

...

 

Results:

...

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

5AL3_ITS_Col3

5AL4_ITS_Col3

5AL5_ITS_Col3

5AL6_ITS_Col3

5AL7_ITS_Col3

5AL8_ITS_Col3

5LVD1_16S_Col3

5LVD2_16S_Col3

Full qPCR results below:

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

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

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

 

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

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

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

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

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

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

5LVD4_16S_Col3

5LVD5_16S_Col3

5LVD1_ITS_Col3

5LVD2_ITS_Col3

5LVD3_ITS_Col3

5LVD4_ITS_Col3

5LVD5_ITS_Col3

5FB1_16S_Col3

Full qPCR results below:

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

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

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

 

04.0002 pM Std

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

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

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

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

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

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

 

 

 

  • 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

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

NTC

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

0.0002 pM Std

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

0.002 pM Std

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

0.02 pM Std

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

0.2 pM Std

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

2 pM Std

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

20 pM Std

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

5FB3_16S_Col3

5FB4_16S_Col3

5FB5_16S_Col3

5FB1_ITS_Col3

5FB2_ITS_Col3

5FB3_ITS_Col3

5FB4_ITS_Col3

5FB5_ITS_Col3

Full qPCR results below:

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

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

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

 

04.0002 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

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

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

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

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

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

 

 

 

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

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

5DG2_16S_Col3

5DG3_16S_Col3

5DG4_16S_Col3

5DG5_16S_Col3

5DG1_ITS_Col3

5DG2_ITS_Col3

5DG3_ITS_Col3

5DG4_ITS_Col3

Full qPCR results below:

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

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

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

0.002 pM Std

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

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

0.2 pM Std

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

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

20 pM Std

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)

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

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

NTC

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

0.0002 pM Std

0.0002 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

0.002 pM Std

0.002 pM Std

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

0.02 pM Std

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

0.2 pM Std

0.2 pM Std

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

2 pM Std

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

20 pM Std

20 pM Std

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

 

 

 

Results:

Plate Name

Average Result (nanomoles)

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

Full qPCR results below:

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:

 

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

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

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

Full qPCR results below: