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5CM1 (16S only)

5CM2 (16S only)

5CM3 (16S only)

5CM4 (16S only)

MasterMix (make for 8 plates in 15mL conical tube)

ul/rxn

Reagent

# of rxns

ul needed (1.5X)

3

5X Kapa HiFi Buffer

768

3456

0.45

10M dNTPs

768

518.4

0.3

Kapa HiFi HotStart DNA Pol

768

345.6

7.25

HPLC H2O

768

8352

11

Total Volume

768

12672

*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 MID

ITS MID

5CM1

16S0A1

N/A

16S0B1

N/A

5CM2

16S0C1

N/A

16S0D1

N/A

5CM3

16S0E1

N/A

16S0F1

N/A

5CM4

16S0G1

N/A

16S0H1

N/A

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_Plate1

  • Make 1:1000 dilutions of column 3 from the PCR plates by adding 1 ul to 999 ul TE in a deep well plate:

 

1

2

3

4

5

6

7

8

9

10

11

12

A

5CM1_16S_Col3

5CM2_16S_Col3

5CM3_16S_Col3

5CM4_16S_Col3

 

NTC

NTC

NTC

B

5CM1_16S_Col3

5CM2_16S_Col3

5CM3_16S_Col3

5CM4_16S_Col3

 

0.0002 pM Std

0.0002 pM Std

0.0002 pM Std

C

5CM1_16S_Col3

5CM2_16S_Col3

5CM3_16S_Col3

5CM4_16S_Col3

 

0.002 pM Std

0.002 pM Std

0.002 pM Std

D

5CM1_16S_Col3

5CM2_16S_Col3

5CM3_16S_Col3

5CM4_16S_Col3

 

0.02 pM Std

0.02 pM Std

0.02 pM Std

E

5CM1_16S_Col3

5CM2_16S_Col3

5CM3_16S_Col3

5CM4_16S_Col3

 

0.2 pM Std

0.2 pM Std

0.2 pM Std

F

5CM1_16S_Col3

5CM2_16S_Col3

5CM3_16S_Col3

5CM4_16S_Col3

 

2 pM Std

2 pM Std

2 pM Std

G

5CM1_16S_Col3

5CM2_16S_Col3

5CM3_16S_Col3

5CM4_16S_Col3

 

20 pM Std

20 pM Std

20 pM Std

H

5CM1_16S_Col3

5CM2_16S_Col3

5CM3_16S_Col3

5CM4_16S_Col3

 

 

 

 

  • Make a 1:1000 dilution of HMax_PP Final Pool to check quality of pippin elution before sending out for RFS.

  • 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)

60

600

2 ul

Primer Premix (10X)

60

120

4 ul

Ultra Pure Water

60

240

16 ul

Total Volume

60

960

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

 

1

2

3

4

5

6

7

8

9

10

11

12

A

5CM1_16S_Col3

5CM2_16S_Col3

5CM3_16S_Col3

5CM4_16S_Col3

HMax_PP_Final

 

NTC

NTC

NTC

B

5CM1_16S_Col3

5CM2_16S_Col3

5CM3_16S_Col3

5CM4_16S_Col3

5RM1_1nm_Pool

 

0.0002 pM Std

0.0002 pM Std

0.0002 pM Std

C

5CM1_16S_Col3

5CM2_16S_Col3

5CM3_16S_Col3

5CM4_16S_Col3

 

0.002 pM Std

0.002 pM Std

0.002 pM Std

D

5CM1_16S_Col3

5CM2_16S_Col3

5CM3_16S_Col3

5CM4_16S_Col3

 

0.02 pM Std

0.02 pM Std

0.02 pM Std

E

5CM1_16S_Col3

5CM2_16S_Col3

5CM3_16S_Col3

5CM4_16S_Col3

 

0.2 pM Std

0.2 pM Std

0.2 pM Std

F

5CM1_16S_Col3

5CM2_16S_Col3

5CM3_16S_Col3

5CM4_16S_Col3

 

2 pM Std

2 pM Std

2 pM Std

G

5CM1_16S_Col3

5CM2_16S_Col3

5CM3_16S_Col3

5CM4_16S_Col3

 

20 pM Std

20 pM Std

20 pM Std

H

5CM1_16S_Col3

5CM2_16S_Col3

5CM3_16S_Col3

5CM4_16S_Col3

 

 

 

 

Results:

Average results for the following plates (column 3):

5CM1_16S:

5CM2_16S:

5CM3_16S:

5CM4_16S:

Results for HMax_PP_Final can be viewed in result report below and have been added to the HMax page here.

Full result report can be viewed below:

Pooling and Shipping:

The client, Chris MacGlover, is concerned about the nasal swabs not sequencing as occurred with his last attempt. We will send 4 nasal and one other sample to be fragment analyzed at CU:

5CM1 A4 (18-143N), 5CM1 H12 (SB33N), 5CM2 A4 ( 18-143T), 5CM3 H12 (51N), and 5CM1 A3 (excontrol)

UWYO Plate Name 

Prev Plate Name 

Well

Sample Name

5CM1

Nasal 1A

A01

blank

5CM1

Nasal 1A

A02

blank

5CM1

Nasal 1A

A03

excontrol

5CM1

Nasal 1A

A04

18-143N

5CM1

Nasal 1A

A05

18-145N

5CM1

Nasal 1A

A06

18-150N

5CM1

Nasal 1A

A07

18-152N

5CM1

Nasal 1A

A08

18-157N

5CM1

Nasal 1A

A09

18-163N

5CM1

Nasal 1A

A10

18-168N

5CM1

Nasal 1A

A11

18-169N

5CM1

Nasal 1A

A12

18-171N

5CM1

Nasal 1A

B01

18-172N

5CM1

Nasal 1A

B02

18-173N

5CM1

Nasal 1A

B03

18-174N

5CM1

Nasal 1A

B04

18-175N

5CM1

Nasal 1A

B05

18-177N

5CM1

Nasal 1A

B06

18-178N

5CM1

Nasal 1A

B07

18-180N

5CM1

Nasal 1A

B08

18-181N

5CM1

Nasal 1A

B09

18-182N

5CM1

Nasal 1A

B10

18-183N

5CM1

Nasal 1A

B11

18-184N

5CM1

Nasal 1A

B12

18-185N

5CM1

Nasal 1A

C01

18-186N

5CM1

Nasal 1A

C02

18-187N

5CM1

Nasal 1A

C03

18-188N

5CM1

Nasal 1A

C04

18-189N

5CM1

Nasal 1A

C05

18-190N

5CM1

Nasal 1A

C06

18-191N

5CM1

Nasal 1A

C07

19-001N

5CM1

Nasal 1A

C08

19-002N

5CM1

Nasal 1A

C09

19-003N

5CM1

Nasal 1A

C10

19-004N

5CM1

Nasal 1A

C11

19-005N

5CM1

Nasal 1A

C12

19-006N

5CM1

Nasal 1A

D01

19-007N

5CM1

Nasal 1A

D02

19-008N

5CM1

Nasal 1A

D03

19-010N

5CM1

Nasal 1A

D04

19-011N

5CM1

Nasal 1A

D05

19-013N

5CM1

Nasal 1A

D06

19-015N

5CM1

Nasal 1A

D07

19-017N

5CM1

Nasal 1A

D08

19-018N

5CM1

Nasal 1A

D09

19-019N

5CM1

Nasal 1A

D10

19-020N

5CM1

Nasal 1A

D11

19-021N

5CM1

Nasal 1A

D12

19-023N

5CM1

Nasal 1A

E01

19-025N

5CM1

Nasal 1A

E02

19-026N

5CM1

Nasal 1A

E03

19-027N

5CM1

Nasal 1A

E04

19-028N

5CM1

Nasal 1A

E05

19-029N

5CM1

Nasal 1A

E06

19-030N

5CM1

Nasal 1A

E07

19-031N

5CM1

Nasal 1A

E08

19-032N

5CM1

Nasal 1A

E09

19-033N

5CM1

Nasal 1A

E10

19-034N

5CM1

Nasal 1A

E11

19-036-N

5CM1

Nasal 1A

E12

19-037N

5CM1

Nasal 1A

F01

19-038N

5CM1

Nasal 1A

F02

19-040N

5CM1

Nasal 1A

F03

19-041N

5CM1

Nasal 1A

F04

19-042N

5CM1

Nasal 1A

F05

19-043N

5CM1

Nasal 1A

F06

19-044N

5CM1

Nasal 1A

F07

19-047N

5CM1

Nasal 1A

F08

19-048N

5CM1

Nasal 1A

F09

19-049N

5CM1

Nasal 1A

F10

19-050N

5CM1

Nasal 1A

F11

19-051N

5CM1

Nasal 1A

F12

19-052N

5CM1

Nasal 1A

G01

19-053N

5CM1

Nasal 1A

G02

19-054N

5CM1

Nasal 1A

G03

19-055N

5CM1

Nasal 1A

G04

19-056N

5CM1

Nasal 1A

G05

19-058N

5CM1

Nasal 1A

G06

19-059N

5CM1

Nasal 1A

G07

19-060N

5CM1

Nasal 1A

G08

19-061N

5CM1

Nasal 1A

G09

19-062N

5CM1

Nasal 1A

G10

19-063N

5CM1

Nasal 1A

G11

19-064N

5CM1

Nasal 1A

G12

19-065N

5CM1

Nasal 1A

H01

C26N

5CM1

Nasal 1A

H02

C27N

5CM1

Nasal 1A

H03

C30N

5CM1

Nasal 1A

H04

C31N

5CM1

Nasal 1A

H05

C32N

5CM1

Nasal 1A

H06

C33N

5CM1

Nasal 1A

H07

C35 N

5CM1

Nasal 1A

H08

SB26N

5CM1

Nasal 1A

H09

SB27N

5CM1

Nasal 1A

H10

SB30N

5CM1

Nasal 1A

H11

SB31N

5CM1

Nasal 1A

H12

SB33N

5CM2

Tonsil 1B

A01

blank

5CM2

Tonsil 1B

A02

blank

5CM2

Tonsil 1B

A03

excontrol

5CM2

Tonsil 1B

A04

18-143T

5CM2

Tonsil 1B

A05

18-145T

5CM2

Tonsil 1B

A06

18-150T

5CM2

Tonsil 1B

A07

18-152T

5CM2

Tonsil 1B

A08

18-154T

5CM2

Tonsil 1B

A09

18-157T

5CM2

Tonsil 1B

A10

18-163T

5CM2

Tonsil 1B

A11

18-168T

5CM2

Tonsil 1B

A12

18-169T

5CM2

Tonsil 1B

B01

18-171T

5CM2

Tonsil 1B

B02

18-172T

5CM2

Tonsil 1B

B03

18-173T

5CM2

Tonsil 1B

B04

18-174T

5CM2

Tonsil 1B

B05

18-175T

5CM2

Tonsil 1B

B06

18-177T

5CM2

Tonsil 1B

B07

18-178T

5CM2

Tonsil 1B

B08

18-180T

5CM2

Tonsil 1B

B09

18-181T

5CM2

Tonsil 1B

B10

18-182T

5CM2

Tonsil 1B

B11

18-183T

5CM2

Tonsil 1B

B12

18-184T

5CM2

Tonsil 1B

C01

18-185T

5CM2

Tonsil 1B

C02

18-186T

5CM2

Tonsil 1B

C03

18-187T

5CM2

Tonsil 1B

C04

18-188T

5CM2

Tonsil 1B

C05

18-189T

5CM2

Tonsil 1B

C06

18-190T

5CM2

Tonsil 1B

C07

18-191T

5CM2

Tonsil 1B

C08

19-001T

5CM2

Tonsil 1B

C09

19-002T

5CM2

Tonsil 1B

C10

19-003T

5CM2

Tonsil 1B

C11

19-004T

5CM2

Tonsil 1B

C12

19-005T

5CM2

Tonsil 1B

D01

19-006T

5CM2

Tonsil 1B

D02

19-007T

5CM2

Tonsil 1B

D03

19-008T

5CM2

Tonsil 1B

D04

19-010T

5CM2

Tonsil 1B

D05

19-011T

5CM2

Tonsil 1B

D06

19-013T

5CM2

Tonsil 1B

D07

19-015T

5CM2

Tonsil 1B

D08

19-017T

5CM2

Tonsil 1B

D09

19-018T

5CM2

Tonsil 1B

D10

19-019T

5CM2

Tonsil 1B

D11

19-020T

5CM2

Tonsil 1B

D12

19-021T

5CM2

Tonsil 1B

E01

19-023T

5CM2

Tonsil 1B

E02

19-025T

5CM2

Tonsil 1B

E03

19-026T

5CM2

Tonsil 1B

E04

19-027T

5CM2

Tonsil 1B

E05

19-028T

5CM2

Tonsil 1B

E06

19-029T

5CM2

Tonsil 1B

E07

19-030T

5CM2

Tonsil 1B

E08

19-031T

5CM2

Tonsil 1B

E09

19-032T

5CM2

Tonsil 1B

E10

19-033T

5CM2

Tonsil 1B

E11

19-034T

5CM2

Tonsil 1B

E12

19-036T

5CM2

Tonsil 1B

F01

19-037T

5CM2

Tonsil 1B

F02

19-038T

5CM2

Tonsil 1B

F03

19-040T

5CM2

Tonsil 1B

F04

19-041T

5CM2

Tonsil 1B

F05

19-042T

5CM2

Tonsil 1B

F06

19-043T

5CM2

Tonsil 1B

F07

19-044T

5CM2

Tonsil 1B

F08

19-047T

5CM2

Tonsil 1B

F09

19-048T

5CM2

Tonsil 1B

F10

19-049T

5CM2

Tonsil 1B

F11

19-050T

5CM2

Tonsil 1B

F12

19-051T

5CM2

Tonsil 1B

G01

19-052T

5CM2

Tonsil 1B

G02

19-053T

5CM2

Tonsil 1B

G03

19-054T

5CM2

Tonsil 1B

G04

19-055T

5CM2

Tonsil 1B

G05

19-056T

5CM2

Tonsil 1B

G06

19-058T

5CM2

Tonsil 1B

G07

19-059T

5CM2

Tonsil 1B

G08

19-060T

5CM2

Tonsil 1B

G09

19-061T

5CM2

Tonsil 1B

G10

19-062T

5CM2

Tonsil 1B

G11

19-063T

5CM2

Tonsil 1B

G12

19-064T

5CM2

Tonsil 1B

H01

19-065T

5CM2

Tonsil 1B

H02

C26T

5CM2

Tonsil 1B

H03

C27T

5CM2

Tonsil 1B

H04

C30T

5CM2

Tonsil 1B

H05

C31T

5CM2

Tonsil 1B

H06

C32T

5CM2

Tonsil 1B

H07

C33T

5CM2

Tonsil 1B

H08

C35 T

5CM2

Tonsil 1B

H09

SB26T

5CM2

Tonsil 1B

H10

SB27T

5CM2

Tonsil 1B

H11

SB30T

5CM2

Tonsil 1B

H12

SB31T

5CM3

N - T 1C

A01

blank

5CM3

N - T 1C

A02

blank

5CM3

N - T 1C

A03

excontrol

5CM3

N - T 1C

A04

SB33T

5CM3

N - T 1C

A05

B12T

5CM3

N - T 1C

A06

B31T

5CM3

N - T 1C

A07

B54T

5CM3

N - T 1C

A08

B60T

5CM3

N - T 1C

A09

BK04T

5CM3

N - T 1C

A10

BK122T

5CM3

N - T 1C

A11

BK128T

5CM3

N - T 1C

A12

BK13T

5CM3

N - T 1C

B01

BK19T

5CM3

N - T 1C

B02

BK20T

5CM3

N - T 1C

B03

BK21T

5CM3

N - T 1C

B04

BK23T

5CM3

N - T 1C

B05

BK24T

5CM3

N - T 1C

B06

BK52T

5CM3

N - T 1C

B07

BK5T

5CM3

N - T 1C

B08

BK83T

5CM3

N - T 1C

B09

BK91T

5CM3

N - T 1C

B10

BK98T

5CM3

N - T 1C

B11

BLU165T

5CM3

N - T 1C

B12

BLU20T

5CM3

N - T 1C

C01

R02T

5CM3

N - T 1C

C02

R03T

5CM3

N - T 1C

C03

R11T

5CM3

N - T 1C

C04

R16T

5CM3

N - T 1C

C05

R18T

5CM3

N - T 1C

C06

R20T

5CM3

N - T 1C

C07

R21T

5CM3

N - T 1C

C08

Y14T

5CM3

N - T 1C

C09

Y46T

5CM3

N - T 1C

C10

Y49T

5CM3

N - T 1C

C11

Y51T

5CM3

N - T 1C

C12

Y52T

5CM3

N - T 1C

D01

Y53T

5CM3

N - T 1C

D02

Y63T

5CM3

N - T 1C

D03

Y66T

5CM3

N - T 1C

D04

Y69T

5CM3

N - T 1C

D05

Y81T

5CM3

N - T 1C

D06

Y84T

5CM3

N - T 1C

D07

Y87T

5CM3

N - T 1C

D08

YA1T

5CM3

N - T 1C

D09

YA3T

5CM3

N - T 1C

D10

FR-Y53AT

5CM3

N - T 1C

D11

FR-Y53T

5CM3

N - T 1C

D12

HG-W120T

5CM3

N - T 1C

E01

19-8349T

5CM3

N - T 1C

E02

19-9053T

5CM3

N - T 1C

E03

B12N

5CM3

N - T 1C

E04

B31N

5CM3

N - T 1C

E05

B54N

5CM3

N - T 1C

E06

B60N

5CM3

N - T 1C

E07

BK04N

5CM3

N - T 1C

E08

BK122N

5CM3

N - T 1C

E09

BK128N

5CM3

N - T 1C

E10

BK13N

5CM3

N - T 1C

E11

BK19N

5CM3

N - T 1C

E12

BK20N

5CM3

N - T 1C

F01

BK21N

5CM3

N - T 1C

F02

BK23N

5CM3

N - T 1C

F03

BK24N

5CM3

N - T 1C

F04

BK52N

5CM3

N - T 1C

F05

BK5N

5CM3

N - T 1C

F06

BK83N

5CM3

N - T 1C

F07

BK91N

5CM3

N - T 1C

F08

BK98N

5CM3

N - T 1C

F09

BLU165N

5CM3

N - T 1C

F10

BLU20N

5CM3

N - T 1C

F11

R02N

5CM3

N - T 1C

F12

R03N

5CM3

N - T 1C

G01

R11N

5CM3

N - T 1C

G02

R16N

5CM3

N - T 1C

G03

R18N

5CM3

N - T 1C

G04

R20N

5CM3

N - T 1C

G05

R21N

5CM3

N - T 1C

G06

Y14N

5CM3

N - T 1C

G07

Y46N

5CM3

N - T 1C

G08

Y49N

5CM3

N - T 1C

G09

Y51N

5CM3

N - T 1C

G10

Y52N

5CM3

N - T 1C

G11

Y53N

5CM3

N - T 1C

G12

Y63N

5CM3

N - T 1C

H01

Y66N

5CM3

N - T 1C

H02

Y69N

5CM3

N - T 1C

H03

Y81N

5CM3

N - T 1C

H04

Y84N

5CM3

N - T 1C

H05

Y87N

5CM3

N - T 1C

H06

YA1N

5CM3

N - T 1C

H07

YA3N

5CM3

N - T 1C

H08

47N

5CM3

N - T 1C

H09

48N

5CM3

N - T 1C

H10

49N

5CM3

N - T 1C

H11

50N

5CM3

N - T 1C

H12

51N

5CM4

Nasal 2D

A01

blank

5CM4

Nasal 2D

A02

blank

5CM4

Nasal 2D

A03

 

5CM4

Nasal 2D

A04

64N

5CM4

Nasal 2D

A05

19-10733N

5CM4

Nasal 2D

A06

19-10734N

5CM4

Nasal 2D

A07

 

5CM4

Nasal 2D

A08

 

5CM4

Nasal 2D

A09

 

5CM4

Nasal 2D

A10

 

5CM4

Nasal 2D

A11

 

5CM4

Nasal 2D

A12

 

5CM4

Nasal 2D

B01

52N

5CM4

Nasal 2D

B02

56N

5CM4

Nasal 2D

B03

60N

5CM4

Nasal 2D

B04

65N

5CM4

Nasal 2D

B05

Y53AN

5CM4

Nasal 2D

B06

19-8349N

5CM4

Nasal 2D

B07

 

5CM4

Nasal 2D

B08

 

5CM4

Nasal 2D

B09

 

5CM4

Nasal 2D

B10

 

5CM4

Nasal 2D

B11

 

5CM4

Nasal 2D

B12

 

5CM4

Nasal 2D

C01

53N

5CM4

Nasal 2D

C02

57N

5CM4

Nasal 2D

C03

61N

5CM4

Nasal 2D

C04

66N

5CM4

Nasal 2D

C05

Y53BN

5CM4

Nasal 2D

C06

19-9053N

5CM4

Nasal 2D

C07

 

5CM4

Nasal 2D

C08

 

5CM4

Nasal 2D

C09

 

5CM4

Nasal 2D

C10

 

5CM4

Nasal 2D

C11

 

5CM4

Nasal 2D

C12

 

5CM4

Nasal 2D

D01

54N

5CM4

Nasal 2D

D02

58N

5CM4

Nasal 2D

D03

62N

5CM4

Nasal 2D

D04

67N

5CM4

Nasal 2D

D05

HG-W120AN

5CM4

Nasal 2D

D06

19-09761N

5CM4

Nasal 2D

D07

 

5CM4

Nasal 2D

D08

 

5CM4

Nasal 2D

D09

 

5CM4

Nasal 2D

D10

 

5CM4

Nasal 2D

D11

 

5CM4

Nasal 2D

D12

 

5CM4

Nasal 2D

E01

55N

5CM4

Nasal 2D

E02

59N

5CM4

Nasal 2D

E03

63N

5CM4

Nasal 2D

E04

68N

5CM4

Nasal 2D

E05

HG-W120BN

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