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: 6.03 nanomoles
5CM2_16S: 9.32 nanomoles
5CM3_16S: 3.52 nanomoles
5CM4_16S: 9.77 nanomoles
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:
| View file | ||
|---|---|---|
|
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
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: 6.03 nanomoles
5CM2_16S: 9.32 nanomoles
5CM3_16S: 3.52 nanomoles
5CM4_16S: 9.77 nanomoles
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:
| View file | ||
|---|---|---|
|
Pooling and Shipping:
The client, Chris MacGlover, is concerned about the nasal swabs not sequencing as occurred with his last attempt. We will send 3 nasal, one excontrol, 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)