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Plates in red have been extracted, PCRed, cleaned, quantified, and normalized by 7-15-
Info
Info

Plates in red have been extracted, PCRed, cleaned, quantified, and normalized by 7-15-22. Bolded red have been qPCRed.

NS6 Plates

6AC1

6AC2

6AC3

6BM1

6BM2

6LVD1

6LVD2

6LVD3

6LVD4

6RD1

6RD2

6RD3

6GTL1

6GTL2

6RD4

6RD5

6RD6

6RD7

6LVD5

6LVD6

6LVD7

MP002

MP003

MP006

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

...

  • 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

...

6LVD5

...

16S0A4

...

ITS0A4

...

16S0B4

...

ITS0B4

...

6LVD6

...

16S0C4

...

ITS0C4

...

16S0D4

...

ITS0D4

...

6LVD7

...

16S0E4

...

ITS0E4

...

16S0F4

...

Plate

16S Primer

ITS Primer

6GTL1

16S0G4

ITS0G4

16S0H4

ITS0H4

6GTL2

16S0A5

ITS0A5

16S0B5

ITS0B5

...

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

 

1

2

3

4

5

6

7

8

9

10

11

12

A

6AC3_ITS

6AC3_16S

6RD1_ITS

6RD1_16S

6BM1_ITS

6BM1_16S

6LVD1_ITS

6LVD1_16S

6GTL1_ITS

6GTL1_16S

20 pM Std

20 pM Std

B

6AC3_ITS

6AC3_16S

6RD1_ITS

6RD1_16S

6BM1_ITS

6BM1_16S

6LVD1_ITS

6LVD1_16S

6GTL1_ITS

6GTL1_16S

2 pM Std

2 pM Std

C

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:

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:

Plate Name

Average Result (nanomoles)

 

1

2

3

4

5

6

7

8

9

10

11

12

A

6AC3_ITS

6AC3_16S

6RD1_ITS

6RD1_16S

6BM1_ITS

6BM1_16S

6LVD1_ITS

6LVD1_16S

6GTL1_ITS

6GTL1_16S

0.2 20 pM Std0.2

20 pM Std

DB

6AC3_ITS

6AC3_16S

6RD1_ITS

6RD1_16S

6BM1_ITS

6BM1_16S

6LVD1_ITS

6LVD1_16S

6GTL1_ITS

6GTL1_16S

0.02 2 pM Std0.02

2 pM Std

EC

6AC3_ITS

6AC3_16S

6RD1_ITS

6RD1_16S

6BM1_ITS

6BM1_16S

6LVD1_ITS

6LVD1_16S

6GTL1_ITS

6GTL1_16S

0.002 2 pM Std

0.002 2 pM Std

FD

6AC3_ITS

6AC3_16S

6RD1_ITS

6RD1_16S

6BM1_ITS

6BM1_16S

6LVD1_ITS

6LVD1_16S

6GTL1_ITS

6GTL1_16S

0.0002 02 pM Std

0.0002 02 pM Std

GE

6AC3_ITS

6AC3_16S

6RD1_ITS

6RD1_16S

6BM1_ITS

6BM1_16S

6LVD1_ITS

6LVD1_16S

6GTL1_ITS

6GTL1_16S

NTC

NTC

H0.002 pM Std

0.002 pM Std

F

6AC3_ITS

6AC3_16S

6RD1_ITS

6RD1_16S

6BM1_ITS

6BM1_16S

6LVD1_ITS

6LVD1_16S

6GTL1_ITS

6GTL1_16S

NTC

NTC

Results:

0.0002 pM Std

0.0002 pM Std

G

6AC3_ITS

4.125

6AC3_16S

7.46

6RD1_ITS

10.152

6RD1_16S

15.709

6BM1_ITS

7.874

6BM1_16S

4.842

6LVD1_ITS

4.532

6LVD1_16S

8.102

6GTL1_ITS

5.992

6GTL1_16S

Needs to be redone

Full qPCR results below:

View file
name6AC3_6RD1_6BM1_6LVD1_6GTL1.csv

qPCR NovaSeq6_Set3

  • Make 1:1000 dilutions of column 8 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

6LVD2_ITS

6LVD2_16S

2TRNL3_16S

2TRNL3_TRNL

2TRNL2_TRNL

2TRNL2_16S

6RD2_16S

6RD2_ITS

6GTL1_16S

2TRNL1_16S_2

 

NTC

B

6LVD2_ITS

6LVD2_16S

2TRNL3_16S

2TRNL3_TRNL

2TRNL2_TRNL

2TRNL2_16S

6RD2_16S

6RD2_ITS

6GTL1_16S

2TRNL1_16S_2

04.0002 pM Std

0.0002 pM Std

C

6LVD2_ITS

6LVD2_16S

2TRNL3_16S

2TRNL3_TRNL

2TRNL2_TRNL

2TRNL2_16S

6RD2_16S

6RD2_ITS

6GTL1_16S

2TRNL1_16S_2

0.002 pM Std

0.002 pM Std

D

NTC

NTC

H

6AC3_ITS

6AC3_16S

6RD1_ITS

6RD1_16S

6BM1_ITS

6BM1_16S

6LVD1_ITS

6LVD1_16S

6GTL1_ITS

6GTL1_16S

NTC

NTC

Results:

Plate Name

Average Result (nanomoles)

6AC3_ITS

4.125

6AC3_16S

7.46

6RD1_ITS

10.152

6RD1_16S

15.709

6BM1_ITS

7.874

6BM1_16S

4.842

6LVD1_ITS

4.532

6LVD1_16S

8.102

6GTL1_ITS

5.992

6GTL1_16S

Needs to be redone

Full qPCR results below:

View file
name6AC3_6RD1_6BM1_6LVD1_6GTL1.csv

qPCR NovaSeq6_Set3

  • Make 1:1000 dilutions of column 8 from the PCR plates by adding 1 ul to 999 ul TE in a deep well plate:

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

 

1

2

3

4

5

6

7

8

9

10

11

12

A

 

1

2

3

4

5

6

7

8

9

10

11

12

A

6LVD2_ITS

6LVD2_16S

2TRNL3_16S

2TRNL3_TRNL

2TRNL2_TRNL

2TRNL2_16S

6RD2_16S

6RD2_ITS

6GTL1_16S

2TRNL1_16S_2

0.02 pM Std

0.02 pM Std

E 

NTC

B

6LVD2_ITS

6LVD2_16S

2TRNL3_16S

2TRNL3_TRNL

2TRNL2_TRNL

2TRNL2_16S

6RD2_16S

6RD2_ITS

6GTL1_16S

2TRNL1_16S_2

004.2 0002 pM Std

0.2 0002 pM Std

FC

6LVD2_ITS

6LVD2_16S

2TRNL3_16S

2TRNL3_TRNL

2TRNL2_TRNL

2TRNL2_16S

6RD2_16S

6RD2_ITS

6GTL1_16S

2TRNL1_16S_2

2 0.002 pM Std

2 0.002 pM Std

GD

6LVD2_ITS

6LVD2_16S

2TRNL3_16S

2TRNL3_TRNL

2TRNL2_TRNL

2TRNL2_16S

6RD2_16S

6RD2_ITS

6GTL1_16S

2TRNL1_16S_2

20 0.02 pM Std

20 0.02 pM Std

HE

6LVD2_ITS

6LVD2_16S

2TRNL3_16S

2TRNL3_TRNL

2TRNL2_TRNL

2TRNL2_16S

6RD2_16S

6RD2_ITS

6GTL1_16S2TRNL1_16S_2

 

 

  • 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

2TRNL1_16S_2

0.2 pM Std

0.2 pM Std

F

6LVD2_ITS

6LVD2_16S

2TRNL3_16S

2TRNL3_TRNL

2TRNL2_TRNL

2TRNL2_16S

6RD2_16S

6RD2_ITS

6GTL1_16S

2TRNL1_16S_2

2 pM Std

2 pM Std

G

6LVD2_ITS

6LVD2_16S

2TRNL3_16S

2TRNL3_TRNL

2TRNL2_TRNL

2TRNL2_16S

6RD2_16S

6RD2_ITS

6GTL1_16S

2TRNL1_16S_2

20 pM Std

20 pM Std

B

H

6LVD2_ITS

6LVD2_16S

2TRNL3_16S

2TRNL3_TRNL

2TRNL2_TRNL

2TRNL2_16S

6RD2_16S

6RD2_ITS

6GTL1_16S

2TRNL1_16S

_2

2 pM Std

2 pM Std

C

6LVD2_ITS

6LVD2_16S

2TRNL3_16S

2TRNL3_TRNL

2TRNL2_TRNL

2TRNL2_16S

6RD2_16S

6RD2_ITS

6GTL1_16S

2TRNL1_16S_2

0.2 pM Std

0.2 pM Std

D

_2

 

 

  • 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:

Plate Name

Average Result (nanomoles)

 

1

2

3

4

5

6

7

8

9

10

11

12

A

6LVD2_ITS

6LVD2_16S

2TRNL3_16S

2TRNL3_TRNL

2TRNL2_TRNL

2TRNL2_16S

6RD2_16S

6RD2_ITS

6GTL1_16S

2TRNL1_16S_2

0.02 20 pM Std0.02

20 pM Std

EB

6LVD2_ITS

6LVD2_16S

2TRNL3_16S

2TRNL3_TRNL

2TRNL2_TRNL

2TRNL2_16S

6RD2_16S

6RD2_ITS

6GTL1_16S

2TRNL1_16S_2

0.002 2 pM Std0.002

2 pM Std

FC

6LVD2_ITS

6LVD2_16S

2TRNL3_16S

2TRNL3_TRNL

2TRNL2_TRNL

2TRNL2_16S

6RD2_16S

6RD2_ITS

6GTL1_16S

2TRNL1_16S_2

0.0002 2 pM Std

0.0002 2 pM Std

GD

6LVD2_ITS

6LVD2_16S

2TRNL3_16S

2TRNL3_TRNL

2TRNL2_TRNL

2TRNL2_16S

6RD2_16S

6RD2_ITS

6GTL1_16S

2TRNL1_16S_2

NTC

NTC

H0.02 pM Std

0.02 pM Std

E

6LVD2_ITS

6LVD2_16S

2TRNL3_16S

2TRNL3_TRNL

2TRNL2_TRNL

2TRNL2_16S

6RD2_16S

6RD2_ITS

6GTL1_16S

2TRNL1_16S_2

NTC

NTC

Results:

0.002 pM Std

0.002 pM Std

F

6LVD2_ITS

16.09

6LVD2_16S

21.71

2TRNL3_16S

42.69

2TRNL3_TRNL

8.68

2TRNL2_TRNL

7.51

2TRNL2_16S

36.27

6RD2_16S

28.48

6RD2_ITS

29.45

6GTL1_16S

8.2

2TRNL1_16S_2

32

0.

72

Full qPCR results below:

...

0002 pM Std

0.0002 pM Std

G

6LVD2_ITS

6LVD2_16S

2TRNL3_16S

2TRNL3_TRNL

2TRNL2_

...

qPCR NovaSeq6_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

...

 

...

 

...

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)

...

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

TRNL

2TRNL2_16S

6RD2_16S

6RD2_ITS

6GTL1_16S

2TRNL1_16S_2

NTC

NTC

H

6LVD2_ITS

6LVD2_16S

2TRNL3_16S

2TRNL3_TRNL

2TRNL2_TRNL

2TRNL2_16S

6RD2_16S

6RD2_ITS

6GTL1_16S

2TRNL1_16S_2

NTC

NTC

Results:

Plate Name

Average Result (nanomoles)

6LVD2_ITS

16.09

6LVD2_16S

21.71

2TRNL3_16S

42.69

2TRNL3_TRNL

8.68

2TRNL2_TRNL

7.51

2TRNL2_16S

36.27

6RD2_16S

28.48

6RD2_ITS

29.45

6GTL1_16S

8.2

2TRNL1_16S_2

32.72

Full qPCR results below:

View file
name6LVD2_2TRNL3_2TRNL2_6RD2_6GTL1_2TRNL1.csv

qPCR NovaSeq6_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

6RD4_16S_Col3

6RD4_ITS_Col3

6RD5_16S_Col3

6RD5_ITS_Col3

6RD6_16S_Col3

6RD6_ITS_Col3

6RD7_16S_Col3

6RD7_ITS_Col3

6LVD5_16S_A1

 

 

NTC

B

6RD4_16S_Col3

6RD4_ITS_Col3

6RD5_16S_Col3

6RD5_ITS_Col3

6RD6_16S_Col3

6RD6_ITS_Col3

6RD7_16S_Col3

6RD7_ITS_Col3

6LVD5_16S_B1

 

04.0002 pM Std

0.0002 pM Std

0.0002 pM Std

C

0.002 pM Std

C

6RD4_16S_Col3

6RD4_ITS_Col3

6RD5_16S_Col3

6RD5_ITS_Col3

6RD6_16S_Col3

6RD6_ITS_Col3

6RD7_16S_Col3

6RD7_ITS_Col3

6LVD5_16S_C1

 

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

6RD4_16S_Col3

6RD4_ITS_Col3

6RD5_16S_Col3

6RD5_ITS_Col3

6RD6_16S_Col3

6RD6_ITS_Col3

6RD7_16S_Col3

6RD7_ITS_Col3

6LVD5_16S_D1

 

0.02 pM Std

0.

2

Full qPCR results below:

qPCR NovaSeq6_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

 

 

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

02 pM Std

F

2 pM Std

2 pM Std

2 pM Std

G

20 pM Std

20 pM Std

20 pM Std

H

 

 

 

Results:

...

Plate Name

...

Average Result (nanomoles)

E

6RD4_16S_Col3

6RD4_ITS_Col3

6RD5_16S_Col3

6RD5_ITS_Col3

6RD6_16S_Col3

6RD6_ITS_Col3

6RD7_16S_Col3

6RD7_ITS_Col3

6LVD5_ITS_A8

 

0.2 pM Std

0.2 pM Std

F

6RD4_16S_Col3

6RD4_ITS_Col3

6RD5_16S_Col3

6RD5_ITS_Col3

6RD6_16S_Col3

6RD6_ITS_Col3

6RD7_16S_Col3

6RD7_ITS_Col3

6LVD5_ITS_B8

 

2 pM Std

2 pM Std

G

6RD4_16S_Col3

6RD4_ITS_Col3

6RD5_16S_Col3

6RD5_ITS_Col3

6RD6_16S_Col3

6RD6_ITS_Col3

6RD7_16S_Col3

6RD7_ITS_Col3

6LVD5_ITS_C8

 

20 pM Std

20 pM Std

H

6RD4_16S_Col3

6RD4_ITS_Col3

6RD5_16S_Col3

6RD5_ITS_Col3

6RD6_16S_Col3

6RD6_ITS_Col3

6RD7_16S_Col3

6RD7_ITS_Col3

6LVD5_ITS_D8

 

 

 

  • 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:

...

Plate Name

...

Average Result (nanomoles)

Full qPCR results below:

qPCR NovaSeq6_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:

...

G

...

20 pM Std

...

20 pM Std

...

20 pM Std

...

H

...

 

...

 

...

 

Results:

...

Plate Name

...

Average Result (nanomoles)

...

 

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

 

11

12

A

6RD4_16S

6RD4_ITS

6RD5_16S

6RD5_ITS

6RD6_16S

6RD6_ITS

6RD7_16S

6RD7_ITS

6LVD5_16S

NTC

NTC

NTC

B

6RD4_16S

6RD4_ITS

6RD5_16S

6RD5_ITS

6RD6_16S

6RD6_ITS

6RD7_16S

6RD7_ITS

6LVD5_16S

0.0002 pM Std

0.0002 pM Std

0.0002 pM Std

C

6RD4_16S

6RD4_ITS

6RD5_16S

6RD5_ITS

6RD6_16S

6RD6_ITS

6RD7_16S

6RD7_ITS

6LVD5_16S

0.002 pM Std

0.002 pM Std

0.002 pM Std

D

6RD4_16S

6RD4_ITS

6RD5_16S

6RD5_ITS

6RD6_16S

6RD6_ITS

6RD7_16S

6RD7_ITS

6LVD5_16S

0.02 pM Std

0.02 pM Std

0.02 pM Std

E

 

0.2 pM Std

0.2 pM Std

F

 

20

6RD4_16S

6RD4_ITS

6RD5_16S

6RD5_ITS

6RD6_16S

6RD6_ITS

6RD7_16S

6RD7_ITS

6LVD5_ITS

0.2 pM Std

0.2 pM Std

G

 

0.2 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

F

6RD4_16S

6RD4_ITS

6RD5_16S

6RD5_ITS

6RD6_16S

6RD6_ITS

6RD7_16S

6RD7_ITS

6LVD5_ITS

2 pM Std

2 pM Std

2 pM Std

G

6RD4_16S

6RD4_ITS

6RD5_16S

6RD5_ITS

6RD6_16S

6RD6_ITS

6RD7_16S

6RD7_ITS

6LVD5_ITS

20 pM Std

20 pM Std

20 pM Std

H

6RD4_16S

6RD4_ITS

6RD5_16S

6RD5_ITS

6RD6_16S

6RD6_ITS

6RD7_16S

6RD7_ITS

6LVD5_ITS

 NS6_Pool_1:1000

  NS6_Pool_1:1000

  NS6_Pool_1:1000

Results:

Plate Name

Average Result (nanomoles)

6RD4_16S

70.66

6RD4_ITS

22.2

6RD5_16S

76.68

6RD5_ITS

86.82

6RD6_16S

32.57

6RD6_ITS

45.66

6RD7_16S

45.71

6RD7_ITS

67.98

6LVD5_16S

50.03

6LVD5_ITS

14.98

NS6_Pool

12.17

Full qPCR results below:

View file
nameNS6_Pool.csv