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95 °C for 60 s, 45 °C for 90 s, 72 °C for 90 s; 28 cycles of: 94 °C for 60 s, 50 °C for 90 s, 72 °C for 60 s

PCR MasterMix

ul/rxn

Reagent

# of rxns

ul needed

7

3

5X Kapa HiFi Buffer

50

150

0.45

10M dNTPs

50

22.5

0.3

Kapa HiFi HotStart DNA

2X

Pol

120

50

900

15

4

7.

5

25

HPLC H2O

120

50

540

362.5

12

11

Total Volume

384

50

1440

550

  • Add 11 ul to each well of a hard shell, full skirt plate. Add 2 uL of 0.5 uM primers and 2uL of template to each well.

  • Primers:

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Step

Temp C

Cycles

Time

Denature

95

1X

10:00

Denature

94

35X

1:00

Annealing** (Row C)

50

35X

1:30

Extension/Elongation

72

35X

1:00

Final Extension

72

1X

10:00

Hold

4

1X

0:00

Pool duplicates together.

MagBead Cleanup:

  • Equilibrate Beads to room Temperature

  • Add 15uL of ultra pure water to each well.

  • Add 24 ul of MagBeads to each well.

  • 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

  • Pool 2 ul of the TRNL and 16S samples separately. Make 1:1000 dilutions of each pool and run in triplicate.

  • Make 1:1000 dilutions of columns 1,6, and 12 for each plate using 999 of TE and 1uL of sample into a deep well plate.

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...

1

...

2

...

3

...

4

...

5

...

6

...

7

...

8

...

9

...

10

...

11

...

12

...

A

...

TRNL1_1_16S_Col1

...

TRNL1_1_16S_Col6

...

TRNL1_1_16S_Col12

...

TRNL1_1_T_Col1

...

TRNL1_1_T_Col6

...

TRNL1_1_T_Col12

...

 

...

NTC

...

NTC

...

NTC

...

B

...

TRNL1_1_16S_Col1

...

TRNL1_1_16S_Col6

...

TRNL1_1_16S_Col12

...

TRNL1_1_T_Col1

...

TRNL1_1_T_Col6

...

TRNL1_1_T_Col12

...

 

...

0.0002 pM Std

...

0.0002 pM Std

...

0.0002 pM Std

...

C

...

TRNL1_1_16S_Col1

...

TRNL1_1_16S_Col6

...

TRNL1_1_16S_Col12

...

TRNL1_1_T_Col1

...

TRNL1_1_T_Col6

...

TRNL1_1_T_Col12

...

 

...

0.002 pM Std

...

0.002 pM Std

...

0.002 pM Std

...

D

...

TRNL1_1_16S_Col1

...

TRNL1_1_16S_Col6

...

TRNL1_1_16S_Col12

...

TRNL1_1_T_Col1

...

TRNL1_1_T_Col6

...

TRNL1_1_T_Col12

...

 

...

0.02 pM Std

...

0.02 pM Std

...

0.02 pM Std

...

E

...

TRNL1_1_16S_Col1

...

TRNL1_1_16S_Col6

...

TRNL1_1_16S_Col12

...

TRNL1_1_T_Col1

...

TRNL1_1_T_Col6

...

TRNL1_1_T_Col12

...

 

...

0.2 pM Std

...

0.2 pM Std

...

0.2 pM Std

...

F

...

TRNL1_1_16S_Col1

...

TRNL1_1_16S_Col6

...

TRNL1_1_16S_Col12

...

TRNL1_1_T_Col1

...

TRNL1_1_T_Col6

...

TRNL1_1_T_Col12

...

 

...

2 pM Std

...

2 pM Std

...

2 pM Std

...

G

...

TRNL1_1_16S_Col1

...

TRNL1_1_16S_Col6

...

TRNL1_1_16S_Col12

...

TRNL1_1_T_Col1

...

TRNL1_1_T_Col6

...

TRNL1_1_T_Col12

...

 

...

20 pM Std

...

20 pM Std

...

20 pM Std

...

H

...

TRNL1_1_16S_Col1

...

TRNL1_1_16S_Col6

...

TRNL1_1_16S_Col12

...

TRNL1_1_T_Col1

...

TRNL1_1_T_Col6

...

TRNL1_1_T_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:

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...

1

...

2

...

3

...

4

...

5

...

6

...

7

...

8

...

9

...

10

...

11

...

12

...

A

...

TRNL1_1_16S_Col1

...

TRNL1_1_16S_Col6

...

TRNL1_1_16S_Col12

...

TRNL1_1_T_Col1

...

TRNL1_1_T_Col6

...

TRNL1_1_T_Col12

...

TRNL1_1_16S_Pool

...

 

...

NTC

...

NTC

...

NTC

...

B

...

TRNL1_1_16S_Col1

...

TRNL1_1_16S_Col6

...

TRNL1_1_16S_Col12

...

TRNL1_1_T_Col1

...

TRNL1_1_T_Col6

...

TRNL1_1_T_Col12

...

TRNL1_1_16S_Pool

...

 

...

0.0002 pM Std

...

0.0002 pM Std

...

0.0002 pM Std

...

C

...

TRNL1_1_16S_Col1

...

TRNL1_1_16S_Col6

...

TRNL1_1_16S_Col12

...

TRNL1_1_T_Col1

...

TRNL1_1_T_Col6

...

TRNL1_1_T_Col12

...

TRNL1_1_16S_Pool

...

 

...

0.002 pM Std

...

0.002 pM Std

...

0.002 pM Std

...

D

...

TRNL1_1_16S_Col1

...

TRNL1_1_16S_Col6

...

TRNL1_1_16S_Col12

...

TRNL1_1_T_Col1

...

TRNL1_1_T_Col6

...

TRNL1_1_T_Col12

...

TRNL1_1_T_Pool

...

 

...

0.02 pM Std

...

0.02 pM Std

...

0.02 pM Std

...

E

...

TRNL1_1_16S_Col1

...

TRNL1_1_16S_Col6

...

TRNL1_1_16S_Col12

...

TRNL1_1_T_Col1

...

TRNL1_1_T_Col6

...

TRNL1_1_T_Col12

...

TRNL1_1_T_Pool

...

 

...

0.2 pM Std

...

0.2 pM Std

...

0.2 pM Std

...

F

...

TRNL1_1_16S_Col1

...

TRNL1_1_16S_Col6

...

TRNL1_1_16S_Col12

...

TRNL1_1_T_Col1

...

TRNL1_1_T_Col6

...

TRNL1_1_T_Col12

...

TRNL1_1_T_Pool

...

 

...

2 pM Std

...

2 pM Std

...

2 pM Std

...

G

...

TRNL1_1_16S_Col1

...

TRNL1_1_16S_Col6

...

TRNL1_1_16S_Col12

...

TRNL1_1_T_Col1

...

TRNL1_1_T_Col6

...

TRNL1_1_T_Col12

...

 

...

20 pM Std

...

20 pM Std

...

20 pM Std

...

H

...

TRNL1_1_16S_Col1

...

TRNL1_1_16S_Col6

...

TRNL1_1_16S_Col12

...

TRNL1_1_T_Col1

...

TRNL1_1_T_Col6

...

TRNL1_1_T_Col12

...

 

...

 

...

 

...

 

Results:

The TRNL pool via standard size estimation returned a mean of ? nM. This should be adjusted for the difference between the standards' fragment sizes and the expected product size (452 vs 220). 9.41x(452/220) = 19.33 nM

16S is close enough to the standards' fragment size that the standard estimation can be used. 16S mean is 46.34 nM.

iSeq Sequencing:

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

  • 1000/Results = ul of Pool to Add

    • 1000/19.33 = 52uL of Pool to Add

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

    • 1000- 52 = 948 uL of 10mM Tris 8.5 to Add

Pool 16S Pool to 1 nM based off qPCR results. qPCR results are in pM, but 1:1000 dilution used. The results are effectively in nM for pool.

  • 1000/Results = ul of Pool to Add

    • 1000/46.34 = 22 uL of Pool to Add

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

    • 1000- 22 = 978 uL of 10mM Tris 8.5

Combine 100 ul TRNL 1 nM pool to 100 ul 16S 1 nM pool to create a combined 1 nM pool

Dilute 1 nM full pool to loading concentration of 50 pM:

  • Add 5 ul 1 nM Pool to 85 ul “10 mM Tris 8.5” and 10 ul 50 pM PhiX

  • Remove iSeq 100 i1 Flow Cell from refrigerator 5’s crisper drawer and open white foil pack and allow to equilibrate to RT for 10-15 minutes.

  • Open “iSeq 100 i1 Reagent Cartridge v2”. Turn on iSeq100

  • Click on “Sequence”. Watch Video. Do what video tells you to do. Follow on screen instructions until run starts.

Run samples on TapeStation:

page1image48924080Image Added

Positive Control Butterfly (D1):

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Extraction Blank(A2):

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Typical Amplification (42/D2):

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Worst Amplification (43/E2):

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Best Amplification (46/H2):

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