NS5 Plates
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* | ||||
5AC1* | 5AC2* | 5AC3* |
*Red plates TBD if they will be on NS5
...
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 “NS5 PCR”. (If doing manually, one needs 2 ul of template and 2 ul of the primers).
...
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 | 4AH1_16S | 4AH1_ITS | 4AH2_16S | 4AH2_ITS | 4PA1_16S | 4PA1_ITS | 4PA2_16S | 4PA2_ITS |
| NTC | NTC | NTC | ||||||||||
B | 4AH1_16S | 4AH1_ITS | 4AH2_16S | 4AH2_ITS | 4PA1_16S | 4PA1_ITS | 4PA2_16S | 4PA2_ITS |
| 0.0002 pM | 0.0002 pM Std | 0.0002 pM Std | 0.0002 pM Std | |||||||||
C4AH1_16S | 4AH1_ITS | 4AH2_16S | 4AH2_ITS | 4PA1_16S | 4PA1_ITS | 4PA2_16S | 4PA2_ITS |
| 0.002 pM Std | 0.002 pM Std | 0.002 pM Std | |||||||||||
D | 4AH1_16S | 4AH1_ITS | 4AH2_16S | 4AH2_ITS | 4PA1_16S | 4PA1_ITS | 4PA2_16S | 4PA2_ITS |
| 0 | 0.02 pM Std | 0.02 pM Std | 0.02 pM Std | |||||||||
E4AH1_16S | 4AH1_ITS | 4AH2_16S | 4AH2_ITS | 4PA1_16S | 4PA1_ITS | 4PA2_16S | 4PA2_ITS |
| 0.2 pM Std | 0.2 pM Std | 0.2 pM Std | |||||||||||
F | 4AH1_16S | 4AH1_ITS | 4AH2_16S | 4AH2_ITS | 4PA1_16S | 4PA1_ITS | 4PA2_16S | 4PA2_ITS |
| 2 pM | 2 pM Std | 2 pM Std | 2 pM Std | |||||||||
G4AH1_16S | 4AH1_ITS | 4AH2_16S | 4AH2_ITS | 4PA1_16S | 4PA1_ITS | 4PA2_16S | 4PA2_ITS |
| 20 pM Std | 20 pM Std | 20 pM Std | |||||||||||
H | 4AH1_16S | 4AH1_ITS | 4AH2_16S | 4AH2_ITS | 4PA1_16S | 4PA1_ITS | 4PA2_16S | 4PA2_ITS |
|
|
|
|
|
|
Add 16 ul of Illumina Library Quantification MasterMix 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 | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
A4AH1_16S | 4AH1_ITS | 4AH2_16S | 4AH2_ITS | 4PA1_16S | 4PA1_ITS | 4PA2_16S | 4PA2_ITS |
| NTC | NTC | NTC | |||||||||||
B | 4AH1_16S | 4AH1_ITS | 4AH2_16S | 4AH2_ITS | 4PA1_16S | 4PA1_ITS | 4PA2_16S | 4PA2_ITS |
| 0. | 0.0002 pM Std | 0.0002 pM Std | 0.0002 pM Std | |||||||||
C4AH1_16S | 4AH1_ITS | 4AH2_16S | 4AH2_ITS | 4PA1_16S | 4PA1_ITS | 4PA2_16S | 4PA2_ITS |
| 0.002 pM Std | 0.002 pM Std | 0.002 pM Std | |||||||||||
D | 4AH1_16S | 4AH1_ITS | 4AH2_16S | 4AH2_ITS | 4PA1_16S | 4PA1_ITS | 4PA2_16S | 4PA2_ITS |
| 0 | 0.02 pM Std | 0.02 pM Std | 0.02 pM Std | |||||||||
E4AH1_16S | 4AH1_ITS | 4AH2_16S | 4AH2_ITS | 4PA1_16S | 4PA1_ITS | 4PA2_16S | 4PA2_ITS |
| 0.2 pM Std | 0.2 pM Std | 0.2 pM Std | |||||||||||
F | 4AH1_16S | 4AH1_ITS | 4AH2_16S | 4AH2_ITS | 4PA1_16S | 4PA1_ITS | 4PA2_16S | 4PA2_ITS |
| 2 pM | 2 pM Std | 2 pM Std | 2 pM Std | |||||||||
G4AH1_16S | 4AH1_ITS | 4AH2_16S | 4AH2_ITS | 4PA1_16S | 4PA1_ITS | 4PA2_16S | 4PA2_ITS |
| 20 pM Std | 20 pM Std | 20 pM Std | |||||||||||
H | 4AH1_16S | 4AH1_ITS | 4AH2_16S | 4AH2_ITS | 4PA1_16S | 4PA1_ITS | 4PA2_16S | 4PA2_ITS |
|
|
|
|
|
|
Results:
Average results for the following plates (column 3):
4AH1_16S: 11.86 nanomoles
4AH1_ITS: 15.74 nanomoles
4AH2_16S: 36.27 nanomoles
4AH2_ITS: 43.5 nanomoles
4PA1_16S: 4.72 nanomoles
4PA1_ITS: 22.76 nanomoles
4PA2_16S: 8.58 nanomoles
4PA2_ITS: 84.66 nanomoles
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:
| View file | ||
|---|---|---|
|
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
...
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 |
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 “NovaSeq4 “NS5 PCR”. (If doing manually, one needs 2 ul of template and 2 ul of the primers).
Run on Thermocycler Program GSAF36:
Temp C
Cycles
Time
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 |
...
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 NovaSeq4_Plate2
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
...
4PA3_16S
...
4PA3_ITS
...
4PA4_16s
...
4PA4_ITS
...
4PA5_16S
...
4PA5_ITS
...
4PA6_16S
...
4PA6_ITS
...
...
...
...
NTC
...
B
...
4PA3_16S
...
4PA3_ITS
...
4PA4_16s
...
4PA4_ITS
...
4PA5_16S
...
4PA5_ITS
...
4PA6_16S
...
4PA6_ITS
...
...
...
04.0002 pM Std
...
0.0002 pM Std
...
C
...
4PA3_16S
...
4PA3_ITS
...
4PA4_16s
...
4PA4_ITS
...
4PA5_16S
...
4PA5_ITS
...
4PA6_16S
...
4PA6_ITS
...
...
...
0.002 pM Std
...
0.002 pM Std
...
D
...
4PA3_16S
...
4PA3_ITS
...
4PA4_16s
...
4PA4_ITS
...
4PA5_16S
...
4PA5_ITS
...
4PA6_16S
...
4PA6_ITS
...
...
...
0.02 pM Std
...
0.02 pM Std
...
E
...
4PA3_16S
...
4PA3_ITS
...
4PA4_16s
...
4PA4_ITS
...
4PA5_16S
...
4PA5_ITS
...
4PA6_16S
...
4PA6_ITS
...
...
...
0.2 pM Std
...
0.2 pM Std
...
F
...
4PA3_16S
...
4PA3_ITS
...
4PA4_16s
...
4PA4_ITS
...
4PA5_16S
...
4PA5_ITS
...
4PA6_16S
...
4PA6_ITS
...
...
...
2 pM Std
...
2 pM Std
...
G
...
4PA3_16S
...
4PA3_ITS
...
4PA4_16s
...
4PA4_ITS
...
4PA5_16S
...
4PA5_ITS
...
4PA6_16S
...
4PA6_ITS
...
...
...
20 pM Std
...
20 pM Std
...
H
...
4PA3_16S
...
4PA3_ITS
...
4PA4_16s
...
4PA4_ITS
...
4PA5_16S
...
4PA5_ITS
...
4PA6_16S
...
4PA6_ITS
...
...
...
...
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
...
4PA3_16S
...
4PA3_ITS
...
4PA4_16s
...
4PA4_ITS
...
4PA5_16S
...
4PA5_ITS
...
4PA6_16S
...
4PA6_ITS
...
...
NTC
...
NTC
...
NTC
...
B
...
4PA3_16S
...
4PA3_ITS
...
4PA4_16s
...
4PA4_ITS
...
4PA5_16S
...
4PA5_ITS
...
4PA6_16S
...
4PA6_ITS
...
...
0.0002 pM Std
...
0.0002 pM Std
...
0.0002 pM Std
...
C
...
4PA3_16S
...
4PA3_ITS
...
4PA4_16s
...
4PA4_ITS
...
4PA5_16S
...
4PA5_ITS
...
4PA6_16S
...
4PA6_ITS
...
...
0.002 pM Std
...
0.002 pM Std
...
0.002 pM Std
...
D
...
4PA3_16S
...
4PA3_ITS
...
4PA4_16s
...
4PA4_ITS
...
4PA5_16S
...
4PA5_ITS
...
4PA6_16S
...
4PA6_ITS
...
...
0.02 pM Std
...
0.02 pM Std
...
0.02 pM Std
...
E
...
4PA3_16S
...
4PA3_ITS
...
4PA4_16s
...
4PA4_ITS
...
4PA5_16S
...
4PA5_ITS
...
4PA6_16S
...
4PA6_ITS
...
...
0.2 pM Std
...
0.2 pM Std
...
0.2 pM Std
...
F
...
4PA3_16S
...
4PA3_ITS
...
4PA4_16s
...
4PA4_ITS
...
4PA5_16S
...
4PA5_ITS
...
4PA6_16S
...
4PA6_ITS
...
...
2 pM Std
...
2 pM Std
...
2 pM Std
...
G
...
4PA3_16S
...
4PA3_ITS
...
4PA4_16s
...
4PA4_ITS
...
4PA5_16S
...
4PA5_ITS
...
4PA6_16S
...
4PA6_ITS
...
...
20 pM Std
...
20 pM Std
...
20 pM Std
...
H
...
4PA3_16S
...
4PA3_ITS
...
4PA4_16s
...
4PA4_ITS
...
4PA5_16S
...
4PA5_ITS
...
4PA6_16S
...
4PA6_ITS
...
...
...
...
Results:
Average results for the following plates (column 3):
4PA3_16S: 4.04 nanomoles
4PA3_ITS: 46.07 nanomoles
4PA4_16S: 71.0 nanomoles
4PA4_ITS: 68.5 nanomoles
4PA5_16S: 10.26 nanomoles
4PA5_ITS: 41.22 nanomoles
4PA6_16S: 11.57 nanomoles
4PA6_ITS: 44.52 nanomoles
Quantities look good for sequencing. The full result report can be viewed below:
| View file | ||
|---|---|---|
|
MasterMix (make for 12 plates in 15mL conical tube)
...
ul/rxn
...
Reagent
...
# of rxns
...
ul needed
...
3
...
5X Kapa HiFi Buffer
...
1210
...
3630
...
0.45
...
10M dNTPs
...
1210
...
544.5
...
0.3
...
Kapa HiFi HotStart DNA Pol
...
1210
...
363
...
7.25
...
HPLC H2O
...
1210
...
8772.5
...
11
...
Total Volume
...
1210
...
13310
Add 11 ul to each well of a hard shell, full skirt plate. Seal with bubble strips and store in refrigerator until needed labeled “NovaSeq4 PCR”. (If doing manually, one needs 2 ul of template and 2 ul of the primers).
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 NovaSeq4_Plate3
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
...
4MR1_16S
...
4MR1_ITS
...
4MR2_16S
...
4MR2_ITS
...
4SC1_16S
...
4SC1_ITS
...
4SC2_16S
...
4SC2_ITS
...
...
...
...
NTC
...
B
...
4MR1_16S
...
4MR1_ITS
...
4MR2_16S
...
4MR2_ITS
...
4SC1_16S
...
4SC1_ITS
...
4SC2_16S
...
4SC2_ITS
...
...
...
04.0002 pM Std
...
0.0002 pM Std
...
C
...
4MR1_16S
...
4MR1_ITS
...
4MR2_16S
...
4MR2_ITS
...
4SC1_16S
...
4SC1_ITS
...
4SC2_16S
...
4SC2_ITS
...
...
...
0.002 pM Std
...
0.002 pM Std
...
D
...
4MR1_16S
...
4MR1_ITS
...
4MR2_16S
...
4MR2_ITS
...
4SC1_16S
...
4SC1_ITS
...
4SC2_16S
...
4SC2_ITS
...
...
...
0.02 pM Std
...
0.02 pM Std
...
E
...
4MR1_16S
...
4MR1_ITS
...
4MR2_16S
...
4MR2_ITS
...
4SC1_16S
...
4SC1_ITS
...
4SC2_16S
...
4SC2_ITS
...
...
...
0.2 pM Std
...
0.2 pM Std
...
F
...
4MR1_16S
...
4MR1_ITS
...
4MR2_16S
...
4MR2_ITS
...
4SC1_16S
...
4SC1_ITS
...
4SC2_16S
...
4SC2_ITS
...
...
...
2 pM Std
...
2 pM Std
...
G
...
4MR1_16S
...
4MR1_ITS
...
4MR2_16S
...
4MR2_ITS
...
4SC1_16S
...
4SC1_ITS
...
4SC2_16S
...
4SC2_ITS
...
...
...
20 pM Std
...
20 pM Std
...
H
...
4MR1_16S
...
4MR1_ITS
...
4MR2_16S
...
4MR2_ITS
...
4SC1_16S
...
4SC1_ITS
...
4SC2_16S
...
4SC2_ITS
...
...
...
...
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
...
4MR1_16S
...
4MR1_ITS
...
4MR2_16S
...
4MR2_ITS
...
4SC1_16S
...
4SC1_ITS
...
4SC2_16S
...
4SC2_ITS
...
...
NTC
...
NTC
...
NTC
...
B
...
4MR1_16S
...
4MR1_ITS
...
4MR2_16S
...
4MR2_ITS
...
4SC1_16S
...
4SC1_ITS
...
4SC2_16S
...
4SC2_ITS
...
...
0.0002 pM Std
...
0.0002 pM Std
...
0.0002 pM Std
...
C
...
4MR1_16S
...
4MR1_ITS
...
4MR2_16S
...
4MR2_ITS
...
4SC1_16S
...
4SC1_ITS
...
4SC2_16S
...
4SC2_ITS
...
...
0.002 pM Std
...
0.002 pM Std
...
0.002 pM Std
...
D
...
4MR1_16S
...
4MR1_ITS
...
4MR2_16S
...
4MR2_ITS
...
4SC1_16S
...
4SC1_ITS
...
4SC2_16S
...
4SC2_ITS
...
...
0.02 pM Std
...
0.02 pM Std
...
0.02 pM Std
...
E
...
4MR1_16S
...
4MR1_ITS
...
4MR2_16S
...
4MR2_ITS
...
4SC1_16S
...
4SC1_ITS
...
4SC2_16S
...
4SC2_ITS
...
...
0.2 pM Std
...
0.2 pM Std
...
0.2 pM Std
...
F
...
4MR1_16S
...
4MR1_ITS
...
4MR2_16S
...
4MR2_ITS
...
4SC1_16S
...
4SC1_ITS
...
4SC2_16S
...
4SC2_ITS
...
...
2 pM Std
...
2 pM Std
...
2 pM Std
...
G
...
4MR1_16S
...
4MR1_ITS
...
4MR2_16S
...
4MR2_ITS
...
4SC1_16S
...
4SC1_ITS
...
4SC2_16S
...
4SC2_ITS
...
...
20 pM Std
...
20 pM Std
...
20 pM Std
...
H
...
4MR1_16S
...
4MR1_ITS
...
4MR2_16S
...
4MR2_ITS
...
4SC1_16S
...
4SC1_ITS
...
4SC2_16S
...
4SC2_ITS
...
...
...
...
Results:
Average results for the following plates (column 3):
4MR1_16S: 92.23 nanomoles
4MR1_ITS: 53.47 nanomoles
4MR2_16S: 105.34 nanomoles
4MR2_ITS: 66.71 nanomoles
4SC1_16S: 46.64 nanomoles
4SC1_ITS: 47.77 nanomoles
4SC2_16S: 65.97 nanomoles
4SC2_ITS: 55.69 nanomoles
Quantities look good for sequencing. The full result report can be viewed below:
| View file | ||
|---|---|---|
|
MasterMix (make for 24 plates in 50mL conical tube)
...
ul/rxn
...
Reagent
...
# of rxns
...
ul needed
...
3
...
5X Kapa HiFi Buffer
...
2500
...
7500
...
0.45
...
10M dNTPs
...
2500
...
1125
...
0.3
...
Kapa HiFi HotStart DNA Pol
...
2500
...
750
...
7.25
...
HPLC H2O
...
2500
...
18125
...
11
...
Total Volume
...
2500
...
27500
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).
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 NovaSeq4_Plate4
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
...
4LVD1_16S
...
4LVD1_ITS
...
4LVD2_16S
...
4LVD2_ITS
...
4LVD3_16S
...
4LVD3_ITS
...
...
...
...
NTC
...
NTC
...
NTC
...
B
...
4LVD1_16S
...
4LVD1_ITS
...
4LVD2_16S
...
4LVD2_ITS
...
4LVD3_16S
...
4LVD3_ITS
...
...
...
...
0.0002 pM Std
...
0.0002 pM Std
...
0.0002 pM Std
...
C
...
4LVD1_16S
...
4LVD1_ITS
...
4LVD2_16S
...
4LVD2_ITS
...
4LVD3_16S
...
4LVD3_ITS
...
...
...
...
0.002 pM Std
...
0.002 pM Std
...
0.002 pM Std
...
D
...
4LVD1_16S
...
4LVD1_ITS
...
4LVD2_16S
...
4LVD2_ITS
...
4LVD3_16S
...
4LVD3_ITS
...
...
...
...
0.02 pM Std
...
0.02 pM Std
...
0.02 pM Std
...
E
...
4LVD1_16S
...
4LVD1_ITS
...
4LVD2_16S
...
4LVD2_ITS
...
4LVD3_16S
...
4LVD3_ITS
...
...
...
...
0.2 pM Std
...
0.2 pM Std
...
0.2 pM Std
...
F
...
4LVD1_16S
...
4LVD1_ITS
...
4LVD2_16S
...
4LVD2_ITS
...
4LVD3_16S
...
4LVD3_ITS
...
...
...
...
2 pM Std
...
2 pM Std
...
2 pM Std
...
G
...
4LVD1_16S
...
4LVD1_ITS
...
4LVD2_16S
...
4LVD2_ITS
...
4LVD3_16S
...
4LVD3_ITS
...
...
...
...
20 pM Std
...
20 pM Std
...
20 pM Std
...
H
...
4LVD1_16S
...
4LVD1_ITS
...
4LVD2_16S
...
4LVD2_ITS
...
4LVD3_16S
...
4LVD3_ITS
...
...
...
...
...
...
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)
...
80
...
800
...
2 ul
...
Primer Premix (10X)
...
80
...
160
...
4 ul
...
Ultra Pure Water
...
80
...
320
...
16 ul
...
Total Volume
...
80
...
1280
Add 4 ul of template, pool, or standards to each well:
...
...
1
...
2
...
3
...
4
...
5
...
6
...
7
...
8
...
9
...
10
...
11
...
12
...
A
...
4LVD1_16S
...
4LVD1_ITS
...
4LVD2_16S
...
4LVD2_ITS
...
4LVD3_16S
...
4LVD3_ITS
...
...
...
...
NTC
...
NTC
...
NTC
...
B
...
4LVD1_16S
...
4LVD1_ITS
...
4LVD2_16S
...
4LVD2_ITS
...
4LVD3_16S
...
4LVD3_ITS
...
...
...
...
0.0002 pM Std
...
0.0002 pM Std
...
0.0002 pM Std
...
C
...
4LVD1_16S
...
4LVD1_ITS
...
4LVD2_16S
...
4LVD2_ITS
...
4LVD3_16S
...
4LVD3_ITS
...
...
...
...
0.002 pM Std
...
0.002 pM Std
...
0.002 pM Std
...
D
...
4LVD1_16S
...
4LVD1_ITS
...
4LVD2_16S
...
4LVD2_ITS
...
4LVD3_16S
...
4LVD3_ITS
...
...
...
...
0.02 pM Std
...
0.02 pM Std
...
0.02 pM Std
...
E
...
4LVD1_16S
...
4LVD1_ITS
...
4LVD2_16S
...
4LVD2_ITS
...
4LVD3_16S
...
4LVD3_ITS
...
...
...
...
0.2 pM Std
...
0.2 pM Std
...
0.2 pM Std
...
F
...
4LVD1_16S
...
4LVD1_ITS
...
4LVD2_16S
...
4LVD2_ITS
...
4LVD3_16S
...
4LVD3_ITS
...
...
...
...
2 pM Std
...
2 pM Std
...
2 pM Std
...
G
...
4LVD1_16S
...
4LVD1_ITS
...
4LVD2_16S
...
4LVD2_ITS
...
4LVD3_16S
...
4LVD3_ITS
...
...
...
...
20 pM Std
...
20 pM Std
...
20 pM Std
...
H
...
4LVD1_16S
...
4LVD1_ITS
...
4LVD2_16S
...
4LVD2_ITS
...
4LVD3_16S
...
4LVD3_ITS
...
...
...
...
...
...
Results:
Average results for the following plates (column 3):
4LVD1_16S: 112.54 nanomoles
4LVD1_ITS: 24.71 nanomoles
4LVD2_16S: 95.57 nanomoles
4LVD2_ITS: 34.28 nanomoles
4LVD3_16S: 112.47 nanomoles
4LVD3_ITS: 101.03 nanomoles
Quantities look good for sequencing. The full result report can be viewed below:
| View file | ||
|---|---|---|
|
qPCR NovaSeq4_Plate5
Make 1:1000 dilutions of column 3 from the PCR plates by adding 1 ul to 999 ul TE in a deep well plate:
Use other half of NS4_Plate 4 dilution plate to do NS4_Plate5 dilution plate.
...
...
1
...
2
...
3
...
4
...
5
...
6
...
7
...
8
...
9
...
10
...
11
...
12
...
A
...
______
...
______
...
______
...
______
...
______
...
______
...
4FB1_16S
...
4FB1_ITS
...
4FB2_16S
...
4FB2_ITS
...
4FB3_16S
...
4FB3_ITS
...
B
...
______
...
______
...
______
...
______
...
______
...
______
...
4FB1_16S
...
4FB1_ITS
...
4FB2_16S
...
4FB2_ITS
...
4FB3_16S
...
4FB3_ITS
...
C
...
______
...
______
...
______
...
______
...
______
...
______
...
4FB1_16S
...
4FB1_ITS
...
4FB2_16S
...
4FB2_ITS
...
4FB3_16S
...
4FB3_ITS
...
D
...
______
...
______
...
______
...
______
...
______
...
______
...
4FB1_16S
...
4FB1_ITS
...
4FB2_16S
...
4FB2_ITS
...
4FB3_16S
...
4FB3_ITS
...
E
...
______
...
______
...
______
...
______
...
______
...
______
...
4FB1_16S
...
4FB1_ITS
...
4FB2_16S
...
4FB2_ITS
...
4FB3_16S
...
4FB3_ITS
...
F
...
______
...
______
...
______
...
______
...
______
...
______
...
4FB1_16S
...
4FB1_ITS
...
4FB2_16S
...
4FB2_ITS
...
4FB3_16S
...
4FB3_ITS
...
G
...
______
...
______
...
______
...
______
...
______
...
______
...
4FB1_16S
...
4FB1_ITS
...
4FB2_16S
...
4FB2_ITS
...
4FB3_16S
...
4FB3_ITS
...
H
...
______
...
______
...
______
...
______
...
______
...
______
...
4FB1_16S
...
4FB1_ITS
...
4FB2_16S
...
4FB2_ITS
...
4FB3_16S
...
4FB3_ITS
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)
...
80
...
800
...
2 ul
...
Primer Premix (10X)
...
80
...
160
...
4 ul
...
Ultra Pure Water
...
80
...
320
...
16 ul
...
Total Volume
...
80
...
1280
Add 4 ul of template, pool, or standards to each well:
...
...
1
...
2
...
3
...
4
...
5
...
6
...
7
...
8
...
9
...
10
...
11
...
12
...
A
...
4FB1_16S
...
4FB1_ITS
...
4FB2_16S
...
4FB2_ITS
...
4FB3_16S
...
4FB3_ITS
...
...
...
...
NTC
...
NTC
...
NTC
...
B
...
4FB1_16S
...
4FB1_ITS
...
4FB2_16S
...
4FB2_ITS
...
4FB3_16S
...
4FB3_ITS
...
...
...
...
0.0002 pM Std
...
0.0002 pM Std
...
0.0002 pM Std
...
C
...
4FB1_16S
...
4FB1_ITS
...
4FB2_16S
...
4FB2_ITS
...
4FB3_16S
...
4FB3_ITS
...
...
...
...
0.002 pM Std
...
0.002 pM Std
...
0.002 pM Std
...
D
...
4FB1_16S
...
4FB1_ITS
...
4FB2_16S
...
4FB2_ITS
...
4FB3_16S
...
4FB3_ITS
...
...
...
...
0.02 pM Std
...
0.02 pM Std
...
0.02 pM Std
...
E
...
4FB1_16S
...
4FB1_ITS
...
4FB2_16S
...
4FB2_ITS
...
4FB3_16S
...
4FB3_ITS
...
...
...
...
0.2 pM Std
...
0.2 pM Std
...
0.2 pM Std
...
F
...
4FB1_16S
...
4FB1_ITS
...
4FB2_16S
...
4FB2_ITS
...
4FB3_16S
...
4FB3_ITS
...
...
...
...
2 pM Std
...
2 pM Std
...
2 pM Std
...
G
...
4FB1_16S
...
4FB1_ITS
...
4FB2_16S
...
4FB2_ITS
...
4FB3_16S
...
4FB3_ITS
...
...
...
...
20 pM Std
...
20 pM Std
...
20 pM Std
...
H
...
4FB1_16S
...
4FB1_ITS
...
4FB2_16S
...
4FB2_ITS
...
4FB3_16S
...
4FB3_ITS
...
...
...
...
...
...
Results:
Average results for the following plates (column 3):
4FB1_16S: 28.57 nanomoles
4FB1_ITS: 45.42 nanomoles
4FB2_16S: 70.82 nanomoles
4FB2_ITS: 71.52 nanomoles
4FB3_16S: 73.04 nanomoles
...
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:
...