...
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 | 5DG5 | |||
5RD1 (2-Step repeat) - click here for the protocol page. | |||||||
Yoon Plate will be re-pooled | |||||||
Current total number of plates for ITS/16S PCR: 20 + 1 plate repeat (sm19-fire)
...
Some plates were cleaned up using the Nimbus platform protocol “AxyPrep MagBead PCR1 No MM”
Manually, it was done:
...
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
...
...
...
...
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:
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
...
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
...
...
NTC
...
NTC
...
NTC
...
B
...
...
0.0002 pM Std
...
0.0002 pM Std
...
0.0002 pM Std
Store in -20 until ready to qPCR
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 “NS5 PCR”. (If doing manually, one needs 2 ul of template and 2 ul of the primers).
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 |
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)
Store in -20 until ready to qPCR.
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 “NS5 PCR”. (If doing manually, one needs 2 ul of template and 2 ul of the primers).
Plate | 16S Primer | ITS Primer |
|---|
5LVD1 |
16S0A3 |
ITS0A3 |
16S0B3 |
ITS0B3 |
5LVD2 |
16S0C3 |
ITS0C3 |
16S0D3 |
ITS0D3 |
5LVD3 |
16S0E3 |
ITS0E3 |
16S0F3 |
ITS0F3 |
5LVD4 |
16S0G3 |
ITS0G3 |
16S0H3 |
ITS0H3 |
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”
...
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_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:
...
Store in -20 until ready to qPCR.
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 “NS5 PCR”. (If doing manually, one needs 2 ul of template and 2 ul of the primers).
Plate | 16S Primer | ITS Primer |
|---|---|---|
5LVD5 | 16S0A4 | ITS0A4 |
16S0B4 | ITS0B4 | |
5FB1 | 16S0C4 | ITS0C4 |
16S0D4 | ITS0D4 | |
5FB2 | 16S0E4 | ITS0E4 |
16S0F4 | ITS0F4 | |
5FB3 | 16S0G4 | ITS0G4 |
16S0H4 | ITS0H4 |
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)
Store in -20 until ready to qPCR.
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 “NS5 PCR”. (If doing manually, one needs 2 ul of template and 2 ul of the primers).
Plate | 16S Primer | ITS Primer |
|---|
5FB4 |
16S0A5 |
ITS0A5 |
16S0B5 |
ITS0B5 |
5FB5 |
16S0C5 |
ITS0C5 |
16S0D5 |
ITS0D5 |
5DG1 |
16S0E5 |
ITS0E5 |
16S0F5 |
ITS0F5 |
5DG2 |
16S0G5 |
ITS0G5 |
16S0H5 |
ITS0H5 |
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:
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
...
5LVD5
...
16S0A4
...
ITS0A4
...
16S0B4
...
ITS0B4
...
5FB1
...
16S0C4
...
ITS0C4
...
16S0D4
...
ITS0D4
...
5FB2
...
16S0E4
...
ITS0E4
...
16S0F4
...
ITS0F4
...
5FB3
...
16S0G4
...
ITS0G4
...
16S0H4
...
ITS0H4
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_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
...
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_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:
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
Store in -20 until ready to qPCR.
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 “NS5 PCR”. (If doing manually, one needs 2 ul of template and 2 ul of the primers).
Plate | 16S Primer | ITS Primer |
|---|---|---|
5DG3 | 16S0A6 | ITS0A6 |
16S0B6 | ITS0B6 | |
5DG4 | 16S0C6 | ITS0C6 |
16S0D6 | ITS0D6 | |
5DG5 (used only one MID plate since there were only 6 columns of samples. Replicate done on a single plate). | 16S0F6` | ITS0F6 |
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)
Store in -20 until ready to qPCR.
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 | 5AL1_16S_Col3 | 5AL2_16S_Col3 | 5AL3_16S_Col3 | 5AL4_16S_Col3 | 5AL5_16S_Col3 | 5AL6_16S_Col3 | 5AL7_16S_Col3 | 5AL8_16S_Col3 | 5AL1_ITS_Col3 | NTC | NTC | NTC |
B | 5AL1_16S_Col3 | 5AL2_16S_Col3 | 5AL3_16S_Col3 | 5AL4_16S_Col3 | 5AL5_16S_Col3 | 5AL6_16S_Col3 | 5AL7_16S_Col3 | 5AL8_16S_Col3 | 5AL1_ITS_Col3 | 0.0002 pM Std | 0.0002 pM Std | 0.0002 pM Std |
C | 5AL1_16S_Col3 | 5AL2_16S_Col3 | 5AL3_16S_Col3 | 5AL4_16S_Col3 | 5AL5_16S_Col3 | 5AL6_16S_Col3 | 5AL7_16S_Col3 | 5AL8_16S_Col3 | 5AL1_ITS_Col3 | 0.002 pM Std | 0.002 pM Std | 0.002 pM Std |
D | 5AL1_16S_Col3 | 5AL2_16S_Col3 | 5AL3_16S_Col3 | 5AL4_16S_Col3 | 5AL5_16S_Col3 | 5AL6_16S_Col3 | 5AL7_16S_Col3 | 5AL8_16S_Col3 | 5AL1_ITS_Col3 | 0.02 pM Std | 0.02 pM Std | 0.02 pM Std |
E | 5AL1_16S_Col3 | 5AL2_16S_Col3 | 5AL3_16S_Col3 | 5AL4_16S_Col3 | 5AL5_16S_Col3 | 5AL6_16S_Col3 | 5AL7_16S_Col3 | 5AL8_16S_Col3 | 5AL1_ITS_Col3 | 0.2 pM Std | 0.2 pM Std | 0.2 pM Std |
F | 5AL1_16S_Col3 | 5AL2_16S_Col3 | 5AL3_16S_Col3 | 5AL4_16S_Col3 | 5AL5_16S_Col3 | 5AL6_16S_Col3 | 5AL7_16S_Col3 | 5AL8_16S_Col3 | 5AL1_ITS_Col3 | 2 pM Std | 2 pM Std | 2 pM Std |
G | 5AL1_16S_Col3 | 5AL2_16S_Col3 | 5AL3_16S_Col3 | 5AL4_16S_Col3 | 5AL5_16S_Col3 | 5AL6_16S_Col3 | 5AL7_16S_Col3 | 5AL8_16S_Col3 | 5AL1_ITS_Col3 | 20 pM Std | 20 pM Std | 20 pM Std |
H | 5AL1_16S_Col3 | 5AL2_16S_Col3 | 5AL3_16S_Col3 | 5AL4_16S_Col3 | 5AL5_16S_Col3 | 5AL6_16S_Col3 | 5AL7_16S_Col3 | 5AL8_16S_Col3 | 5AL1_ITS_Col3 |
|
|
|
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 | 5AL1_16S_Col3 | 5AL2_16S_Col3 | 5AL3_16S_Col3 | 5AL4_16S_Col3 | 5AL5_16S_Col3 | 5AL6_16S_Col3 | 5AL7_16S_Col3 | 5AL8_16S_Col3 | 5AL1_ITS_Col3 | NTC | NTC | NTC |
B | 5AL1_16S_Col3 | 5AL2_16S_Col3 | 5AL3_16S_Col3 | 5AL4_16S_Col3 | 5AL5_16S_Col3 | 5AL6_16S_Col3 | 5AL7_16S_Col3 | 5AL8_16S_Col3 | 5AL1_ITS_Col3 | 0.0002 pM Std | 0.0002 pM Std | 0.0002 pM Std |
C | 5AL1_16S_Col3 | 5AL2_16S_Col3 | 5AL3_16S_Col3 | 5AL4_16S_Col3 | 5AL5_16S_Col3 | 5AL6_16S_Col3 | 5AL7_16S_Col3 | 5AL8_16S_Col3 | 5AL1_ITS_Col3 | 0.002 pM Std | 0.002 pM Std | 0.002 pM Std |
D |
5AL1_16S_Col3 | 5AL2_16S_Col3 | 5AL3_16S_Col3 | 5AL4_16S_Col3 | 5AL5_16S_Col3 | 5AL6_16S_Col3 | 5AL7_16S_Col3 | 5AL8_16S_Col3 | 5AL1_ITS_Col3 | 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:
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 “NS5 PCR”. (If doing manually, one needs 2 ul of template and 2 ul of the primers).
...
Plate
...
16S Primer
...
ITS Primer
...
5FB4
...
16S0A5
...
ITS0A5
...
16S0B5
...
ITS0B5
...
5FB5
...
16S0C5
...
ITS0C5
...
16S0D5
...
ITS0D5
...
5DG1
...
16S0E5
...
ITS0E5
...
16S0F5
...
ITS0F5
...
5DG2
...
16S0G5
...
ITS0G5
...
16S0H5
...
ITS0H5
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_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
...
5AL1_16S_Col3 | 5AL2_16S_Col3 | 5AL3_16S_Col3 | 5AL4_16S_Col3 | 5AL5_16S_Col3 | 5AL6_16S_Col3 | 5AL7_16S_Col3 | 5AL8_16S_Col3 | 5AL1_ITS_Col3 | 0.2 pM Std | 0.2 pM Std | 0.2 pM Std | |
F | 5AL1_16S_Col3 | 5AL2_16S_Col3 | 5AL3_16S_Col3 | 5AL4_16S_Col3 | 5AL5_16S_Col3 | 5AL6_16S_Col3 | 5AL7_16S_Col3 | 5AL8_16S_Col3 | 5AL1_ITS_Col3 | 2 pM Std | 2 pM Std | 2 pM Std |
G | 5AL1_16S_Col3 | 5AL2_16S_Col3 | 5AL3_16S_Col3 | 5AL4_16S_Col3 | 5AL5_16S_Col3 | 5AL6_16S_Col3 | 5AL7_16S_Col3 | 5AL8_16S_Col3 | 5AL1_ITS_Col3 | 20 pM Std | 20 pM Std | 20 pM Std |
H | 5AL1_16S_Col3 | 5AL2_16S_Col3 | 5AL3_16S_Col3 | 5AL4_16S_Col3 | 5AL5_16S_Col3 | 5AL6_16S_Col3 | 5AL7_16S_Col3 | 5AL8_16S_Col3 | 5AL1_ITS_Col3 |
|
|
|
Results:
Plate Name | Average Result (nanomoles) |
|---|---|
5AL1_16S_Col3 | 15.27 |
5AL2_16S_Col3 | 8.38 |
5AL3_16S_Col3 | 20.55 |
5AL4_16S_Col3 | 59.48 |
5AL5_16S_Col3 | 18.49 |
5AL6_16S_Col3 | 1.33 |
5AL7_16S_Col3 | 10.39 |
5AL8_16S_Col3 | 11.29 |
5AL1_ITS_Col3 | 68.89 |
Full qPCR results below:
| View file | ||
|---|---|---|
|
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 | 5AL2_ITS_Col3 | 5AL3_ITS_Col3 | 5AL4_ITS_Col3 | 5AL5_ITS_Col3 | 5AL6_ITS_Col3 | 5AL7_ITS_Col3 | 5AL8_ITS_Col3 | 5LVD1_16S_Col3 | 5LVD2_16S_Col3 |
|
| NTC |
B | 5AL2_ITS_Col3 | 5AL3_ITS_Col3 | 5AL4_ITS_Col3 | 5AL5_ITS_Col3 | 5AL6_ITS_Col3 | 5AL7_ITS_Col3 | 5AL8_ITS_Col3 | 5LVD1_16S_Col3 | 5LVD2_16S_Col3 |
| 04.0002 pM Std | 0.0002 pM Std |
C | 5AL2_ITS_Col3 | 5AL3_ITS_Col3 | 5AL4_ITS_Col3 | 5AL5_ITS_Col3 | 5AL6_ITS_Col3 | 5AL7_ITS_Col3 | 5AL8_ITS_Col3 | 5LVD1_16S_Col3 | 5LVD2_16S_Col3 |
| 0.002 pM Std | 0.002 pM Std |
D | 5AL2_ITS_Col3 | 5AL3_ITS_Col3 | 5AL4_ITS_Col3 | 5AL5_ITS_Col3 | 5AL6_ITS_Col3 | 5AL7_ITS_Col3 | 5AL8_ITS_Col3 | 5LVD1_16S_Col3 | 5LVD2_16S_Col3 |
| 0.02 pM Std | 0.02 pM Std |
E | 5AL2_ITS_Col3 | 5AL3_ITS_Col3 | 5AL4_ITS_Col3 | 5AL5_ITS_Col3 | 5AL6_ITS_Col3 | 5AL7_ITS_Col3 | 5AL8_ITS_Col3 | 5LVD1_16S_Col3 | 5LVD2_16S_Col3 |
| 0.2 pM Std | 0.2 pM Std |
F | 5AL2_ITS_Col3 | 5AL3_ITS_Col3 | 5AL4_ITS_Col3 | 5AL5_ITS_Col3 | 5AL6_ITS_Col3 | 5AL7_ITS_Col3 | 5AL8_ITS_Col3 | 5LVD1_16S_Col3 | 5LVD2_16S_Col3 |
| 2 pM Std | 2 pM Std |
G | 5AL2_ITS_Col3 | 5AL3_ITS_Col3 | 5AL4_ITS_Col3 | 5AL5_ITS_Col3 | 5AL6_ITS_Col3 | 5AL7_ITS_Col3 | 5AL8_ITS_Col3 | 5LVD1_16S_Col3 | 5LVD2_16S_Col3 |
| 20 pM Std | 20 pM Std |
H | 5AL2_ITS_Col3 | 5AL3_ITS_Col3 | 5AL4_ITS_Col3 | 5AL5_ITS_Col3 | 5AL6_ITS_Col3 | 5AL7_ITS_Col3 | 5AL8_ITS_Col3 | 5LVD1_16S_Col3 | 5LVD2_16S_Col3 |
|
|
|
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 | 5AL2_ITS_Col3 | 5AL3_ITS_Col3 | 5AL4_ITS_Col3 | 5AL5_ITS_Col3 | 5AL6_ITS_Col3 | 5AL7_ITS_Col3 | 5AL8_ITS_Col3 | 5LVD1_16S_Col3 | 5LVD2_16S_Col3 | NTC | NTC | NTC |
B | 5AL2_ITS_Col3 | 5AL3_ITS_Col3 | 5AL4_ITS_Col3 | 5AL5_ITS_Col3 | 5AL6_ITS_Col3 | 5AL7_ITS_Col3 | 5AL8_ITS_Col3 | 5LVD1_16S_Col3 | 5LVD2_16S_Col3 | 0.0002 pM Std | 0.0002 pM Std | 0.0002 pM Std |
C | 5AL2_ITS_Col3 | 5AL3_ITS_Col3 | 5AL4_ITS_Col3 | 5AL5_ITS_Col3 | 5AL6_ITS_Col3 | 5AL7_ITS_Col3 | 5AL8_ITS_Col3 | 5LVD1_16S_Col3 | 5LVD2_16S_Col3 | 0.002 pM Std | 0.002 pM Std | 0.002 pM Std |
D | 5AL2_ITS_Col3 | 5AL3_ITS_Col3 | 5AL4_ITS_Col3 | 5AL5_ITS_Col3 | 5AL6_ITS_Col3 | 5AL7_ITS_Col3 | 5AL8_ITS_Col3 | 5LVD1_16S_Col3 | 5LVD2_16S_Col3 | 0.02 pM Std | 0.02 pM Std | 0.02 pM Std |
E | 5AL2_ITS_Col3 | 5AL3_ITS_Col3 | 5AL4_ITS_Col3 | 5AL5_ITS_Col3 | 5AL6_ITS_Col3 | 5AL7_ITS_Col3 | 5AL8_ITS_Col3 | 5LVD1_16S_Col3 | 5LVD2_16S_Col3 | 0.2 pM Std | 0.2 pM Std | 0.2 pM Std |
F | 5AL2_ITS_Col3 | 5AL3_ITS_Col3 | 5AL4_ITS_Col3 | 5AL5_ITS_Col3 | 5AL6_ITS_Col3 | 5AL7_ITS_Col3 | 5AL8_ITS_Col3 | 5LVD1_16S_Col3 | 5LVD2_16S_Col3 | 2 pM Std | 2 pM Std | 2 pM Std |
G | 5AL2_ITS_Col3 | 5AL3_ITS_Col3 | 5AL4_ITS_Col3 | 5AL5_ITS_Col3 | 5AL6_ITS_Col3 | 5AL7_ITS_Col3 | 5AL8_ITS_Col3 | 5LVD1_16S_Col3 | 5LVD2_16S_Col3 | 20 pM Std | 20 pM Std | 20 pM Std |
H | 5AL2_ITS_Col3 | 5AL3_ITS_Col3 | 5AL4_ITS_Col3 | 5AL5_ITS_Col3 | 5AL6_ITS_Col3 | 5AL7_ITS_Col3 | 5AL8_ITS_Col3 | 5LVD1_16S_Col3 | 5LVD2_16S_Col3 |
|
|
|
Results:
Plate Name | Average Result (nanomoles) |
|---|---|
5AL2_ITS_Col3 | 23.49 |
5AL3_ITS_Col3 | 25.91 |
5AL4_ITS_Col3 | 28.89 |
5AL5_ITS_Col3 | 37.02 |
5AL6_ITS_Col3 | 18.74 |
5AL7_ITS_Col3 | 30.43 |
5AL8_ITS_Col3 | 14.8 |
5LVD1_16S_Col3 | 61.73 |
5LVD2_16S_Col3 | 57.63 |
Full qPCR results below:
| View file | ||
|---|---|---|
|
qPCR NovaSeq5_Set3
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 | 5LVD3_16S_Col3 | 5LVD4_16S_Col3 | 5LVD5_16S_Col3 | 5LVD1_ITS_Col3 | 5LVD2_ITS_Col3 | 5LVD3_ITS_Col3 | 5LVD4_ITS_Col3 | 5LVD5_ITS_Col3 | 5FB1_16S_Col3 |
|
| NTC |
B | 5LVD3_16S_Col3 | 5LVD4_16S_Col3 | 5LVD5_16S_Col3 | 5LVD1_ITS_Col3 | 5LVD2_ITS_Col3 | 5LVD3_ITS_Col3 | 5LVD4_ITS_Col3 | 5LVD5_ITS_Col3 | 5FB1_16S_Col3 |
| 04.0002 pM Std | 0.0002 pM Std |
C | 5LVD3_16S_Col3 | 5LVD4_16S_Col3 | 5LVD5_16S_Col3 | 5LVD1_ITS_Col3 | 5LVD2_ITS_Col3 | 5LVD3_ITS_Col3 | 5LVD4_ITS_Col3 | 5LVD5_ITS_Col3 | 5FB1_16S_Col3 |
| 0.002 pM Std | 0.002 pM Std |
D | 5LVD3_16S_Col3 | 5LVD4_16S_Col3 | 5LVD5_16S_Col3 | 5LVD1_ITS_Col3 | 5LVD2_ITS_Col3 | 5LVD3_ITS_Col3 | 5LVD4_ITS_Col3 | 5LVD5_ITS_Col3 | 5FB1_16S_Col3 |
| 0.02 pM Std | 0.02 pM Std |
E | 5LVD3_16S_Col3 | 5LVD4_16S_Col3 | 5LVD5_16S_Col3 | 5LVD1_ITS_Col3 | 5LVD2_ITS_Col3 | 5LVD3_ITS_Col3 | 5LVD4_ITS_Col3 | 5LVD5_ITS_Col3 | 5FB1_16S_Col3 |
| 0.2 pM Std | 0.2 pM Std |
F | 5LVD3_16S_Col3 | 5LVD4_16S_Col3 | 5LVD5_16S_Col3 | 5LVD1_ITS_Col3 | 5LVD2_ITS_Col3 | 5LVD3_ITS_Col3 | 5LVD4_ITS_Col3 | 5LVD5_ITS_Col3 | 5FB1_16S_Col3 |
| 2 pM Std | 2 pM Std |
G | 5LVD3_16S_Col3 | 5LVD4_16S_Col3 | 5LVD5_16S_Col3 | 5LVD1_ITS_Col3 | 5LVD2_ITS_Col3 | 5LVD3_ITS_Col3 | 5LVD4_ITS_Col3 | 5LVD5_ITS_Col3 | 5FB1_16S_Col3 |
| 20 pM Std | 20 pM Std |
H | 5LVD3_16S_Col3 | 5LVD4_16S_Col3 | 5LVD5_16S_Col3 | 5LVD1_ITS_Col3 | 5LVD2_ITS_Col3 | 5LVD3_ITS_Col3 | 5LVD4_ITS_Col3 | 5LVD5_ITS_Col3 | 5FB1_16S_Col3 |
|
|
|
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 | 5LVD3_16S_Col3 | 5LVD4_16S_Col3 | 5LVD5_16S_Col3 | 5LVD1_ITS_Col3 | 5LVD2_ITS_Col3 | 5LVD3_ITS_Col3 | 5LVD4_ITS_Col3 | 5LVD5_ITS_Col3 | 5FB1_16S_Col3 | NTC | NTC | NTC |
B | 5LVD3_16S_Col3 | 5LVD4_16S_Col3 | 5LVD5_16S_Col3 | 5LVD1_ITS_Col3 | 5LVD2_ITS_Col3 | 5LVD3_ITS_Col3 | 5LVD4_ITS_Col3 | 5LVD5_ITS_Col3 | 5FB1_16S_Col3 | 0.0002 pM Std | 0.0002 pM Std | 0.0002 pM Std |
C | 5LVD3_16S_Col3 | 5LVD4_16S_Col3 | 5LVD5_16S_Col3 | 5LVD1_ITS_Col3 | 5LVD2_ITS_Col3 | 5LVD3_ITS_Col3 | 5LVD4_ITS_Col3 | 5LVD5_ITS_Col3 | 5FB1_16S_Col3 | 0.002 pM Std | 0.002 pM Std | 0.002 pM Std |
D | 5LVD3_16S_Col3 | 5LVD4_16S_Col3 | 5LVD5_16S_Col3 | 5LVD1_ITS_Col3 | 5LVD2_ITS_Col3 | 5LVD3_ITS_Col3 | 5LVD4_ITS_Col3 | 5LVD5_ITS_Col3 | 5FB1_16S_Col3 | 0.02 pM Std | 0.02 pM Std | 0.02 pM Std |
E | 5LVD3_16S_Col3 | 5LVD4_16S_Col3 | 5LVD5_16S_Col3 | 5LVD1_ITS_Col3 | 5LVD2_ITS_Col3 | 5LVD3_ITS_Col3 | 5LVD4_ITS_Col3 | 5LVD5_ITS_Col3 | 5FB1_16S_Col3 | 0.2 pM Std | 0.2 pM Std | 0.2 pM Std |
F | 5LVD3_16S_Col3 | 5LVD4_16S_Col3 | 5LVD5_16S_Col3 | 5LVD1_ITS_Col3 | 5LVD2_ITS_Col3 | 5LVD3_ITS_Col3 | 5LVD4_ITS_Col3 | 5LVD5_ITS_Col3 | 5FB1_16S_Col3 | 2 pM Std | 2 pM Std | 2 pM Std |
G | 5LVD3_16S_Col3 | 5LVD4_16S_Col3 | 5LVD5_16S_Col3 | 5LVD1_ITS_Col3 | 5LVD2_ITS_Col3 | 5LVD3_ITS_Col3 | 5LVD4_ITS_Col3 | 5LVD5_ITS_Col3 | 5FB1_16S_Col3 | 20 pM Std | 20 pM Std | 20 pM Std |
H | 5LVD3_16S_Col3 | 5LVD4_16S_Col3 | 5LVD5_16S_Col3 | 5LVD1_ITS_Col3 | 5LVD2_ITS_Col3 | 5LVD3_ITS_Col3 | 5LVD4_ITS_Col3 | 5LVD5_ITS_Col3 | 5FB1_16S_Col3 |
|
|
|
Results:
Plate Name | Average Result (nanomoles) |
|---|---|
5LVD3_16S_Col3 | 60.49 |
5LVD4_16S_Col3 | 56.58 |
5LVD5_16S_Col3 | 59.58 |
5LVD1_ITS_Col3 | 52.72 |
5LVD2_ITS_Col3 | 25.43 |
5LVD3_ITS_Col3 | 39.91 |
5LVD4_ITS_Col3 | 38.61 |
5LVD5_ITS_Col3 | 21.75 |
5FB1_16S_Col3 | 66.78 |
Full qPCR results below:
| View file | ||
|---|---|---|
|
qPCR NovaSeq5_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 | 5FB2_16S_Col3 | 5FB3_16S_Col3 | 5FB4_16S_Col3 | 5FB5_16S_Col3 | 5FB1_ITS_Col3 | 5FB2_ITS_Col3 | 5FB3_ITS_Col3 | 5FB4_ITS_Col3 | 5FB5_ITS_Col3 |
|
| NTC |
B | 5FB2_16S_Col3 | 5FB3_16S_Col3 | 5FB4_16S_Col3 | 5FB5_16S_Col3 | 5FB1_ITS_Col3 | 5FB2_ITS_Col3 | 5FB3_ITS_Col3 | 5FB4_ITS_Col3 | 5FB5_ITS_Col3 |
| 04.0002 pM Std | 0.0002 pM Std |
C | 5FB2_16S_Col3 | 5FB3_16S_Col3 | 5FB4_16S_Col3 | 5FB5_16S_Col3 | 5FB1_ITS_Col3 | 5FB2_ITS_Col3 | 5FB3_ITS_Col3 | 5FB4_ITS_Col3 | 5FB5_ITS_Col3 |
| 0.002 pM Std | 0.002 pM Std |
D | 5FB2_16S_Col3 | 5FB3_16S_Col3 | 5FB4_16S_Col3 | 5FB5_16S_Col3 | 5FB1_ITS_Col3 | 5FB2_ITS_Col3 | 5FB3_ITS_Col3 | 5FB4_ITS_Col3 | 5FB5_ITS_Col3 |
| 0.02 pM Std | 0.02 pM Std |
E | 5FB2_16S_Col3 | 5FB3_16S_Col3 | 5FB4_16S_Col3 | 5FB5_16S_Col3 | 5FB1_ITS_Col3 | 5FB2_ITS_Col3 | 5FB3_ITS_Col3 | 5FB4_ITS_Col3 | 5FB5_ITS_Col3 |
| 0.2 pM Std | 0.2 pM Std |
F | 5FB2_16S_Col3 | 5FB3_16S_Col3 | 5FB4_16S_Col3 | 5FB5_16S_Col3 | 5FB1_ITS_Col3 | 5FB2_ITS_Col3 | 5FB3_ITS_Col3 | 5FB4_ITS_Col3 | 5FB5_ITS_Col3 |
| 2 pM Std | 2 pM Std |
G | 5FB2_16S_Col3 | 5FB3_16S_Col3 | 5FB4_16S_Col3 | 5FB5_16S_Col3 | 5FB1_ITS_Col3 | 5FB2_ITS_Col3 | 5FB3_ITS_Col3 | 5FB4_ITS_Col3 | 5FB5_ITS_Col3 |
| 20 pM Std | 20 pM Std |
H | 5FB2_16S_Col3 | 5FB3_16S_Col3 | 5FB4_16S_Col3 | 5FB5_16S_Col3 | 5FB1_ITS_Col3 | 5FB2_ITS_Col3 | 5FB3_ITS_Col3 | 5FB4_ITS_Col3 | 5FB5_ITS_Col3 |
|
|
|
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 | 5FB2_16S_Col3 | 5FB3_16S_Col3 | 5FB4_16S_Col3 | 5FB5_16S_Col3 | 5FB1_ITS_Col3 | 5FB2_ITS_Col3 | 5FB3_ITS_Col3 | 5FB4_ITS_Col3 | 5FB5_ITS_Col3 | NTC | NTC | NTC |
B | 5FB2_16S_Col3 | 5FB3_16S_Col3 | 5FB4_16S_Col3 | 5FB5_16S_Col3 | 5FB1_ITS_Col3 | 5FB2_ITS_Col3 | 5FB3_ITS_Col3 | 5FB4_ITS_Col3 | 5FB5_ITS_Col3 | 0.0002 pM Std | 0.0002 pM Std | 0.0002 pM Std |
C | 5FB2_16S_Col3 | 5FB3_16S_Col3 | 5FB4_16S_Col3 | 5FB5_16S_Col3 | 5FB1_ITS_Col3 | 5FB2_ITS_Col3 | 5FB3_ITS_Col3 | 5FB4_ITS_Col3 | 5FB5_ITS_Col3 | 0.002 pM Std | 0.002 pM Std | 0.002 pM Std |
D | 5FB2_16S_Col3 | 5FB3_16S_Col3 | 5FB4_16S_Col3 | 5FB5_16S_Col3 | 5FB1_ITS_Col3 | 5FB2_ITS_Col3 | 5FB3_ITS_Col3 | 5FB4_ITS_Col3 | 5FB5_ITS_Col3 | 0.02 pM Std | 0.02 pM Std | 0.02 pM Std |
E | 5FB2_16S_Col3 | 5FB3_16S_Col3 | 5FB4_16S_Col3 | 5FB5_16S_Col3 | 5FB1_ITS_Col3 | 5FB2_ITS_Col3 | 5FB3_ITS_Col3 | 5FB4_ITS_Col3 | 5FB5_ITS_Col3 | 0.2 pM Std | 0.2 pM Std | 0.2 pM Std |
F | 5FB2_16S_Col3 | 5FB3_16S_Col3 | 5FB4_16S_Col3 | 5FB5_16S_Col3 | 5FB1_ITS_Col3 | 5FB2_ITS_Col3 | 5FB3_ITS_Col3 | 5FB4_ITS_Col3 | 5FB5_ITS_Col3 | 2 pM Std | 2 pM Std | 2 pM Std |
G | 5FB2_16S_Col3 | 5FB3_16S_Col3 | 5FB4_16S_Col3 | 5FB5_16S_Col3 | 5FB1_ITS_Col3 | 5FB2_ITS_Col3 | 5FB3_ITS_Col3 | 5FB4_ITS_Col3 | 5FB5_ITS_Col3 | 20 pM Std | 20 pM Std | 20 pM Std |
H | 5FB2_16S_Col3 | 5FB3_16S_Col3 | 5FB4_16S_Col3 | 5FB5_16S_Col3 | 5FB1_ITS_Col3 | 5FB2_ITS_Col3 | 5FB3_ITS_Col3 | 5FB4_ITS_Col3 | 5FB5_ITS_Col3 |
|
|
|
Results:
Plate Name | Average Result (nanomoles) |
|---|---|
5FB2_16S_Col3 | 35.64 |
5FB3_16S_Col3 | 31.55 |
5FB4_16S_Col3 | 45.58 |
5FB5_16S_Col3 | 9.5 |
5FB1_ITS_Col3 | 19.46 |
5FB2_ITS_Col3 | 83.23 |
5FB3_ITS_Col3 | 59.86 |
5FB4_ITS_Col3 | 45.49 |
5FB5_ITS_Col3 | 16.69 |
Full qPCR results below:
| View file | ||
|---|---|---|
|
qPCR NovaSeq5_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 | 5DG1_16S_Col3 | 5DG2_16S_Col3 | 5DG3_16S_Col3 | 5DG4_16S_Col3 | 5DG5_16S_Col3 | 5DG1_ITS_Col3 | 5DG2_ITS_Col3 | 5DG3_ITS_Col3 | 5DG4_ITS_Col3 |
|
| NTC |
B | 5DG1_16S_Col3 | 5DG2_16S_Col3 | 5DG3_16S_Col3 | 5DG4_16S_Col3 | 5DG5_16S_Col3 | 5DG1_ITS_Col3 | 5DG2_ITS_Col3 | 5DG3_ITS_Col3 | 5DG4_ITS_Col3 |
| 04.0002 pM Std | 0.0002 pM Std |
C | 5DG1_16S_Col3 | 5DG2_16S_Col3 | 5DG3_16S_Col3 | 5DG4_16S_Col3 | 5DG5_16S_Col3 | 5DG1_ITS_Col3 | 5DG2_ITS_Col3 | 5DG3_ITS_Col3 | 5DG4_ITS_Col3 |
| 0.002 pM Std | 0.002 pM Std |
D | 5DG1_16S_Col3 | 5DG2_16S_Col3 | 5DG3_16S_Col3 | 5DG4_16S_Col3 | 5DG5_16S_Col3 | 5DG1_ITS_Col3 | 5DG2_ITS_Col3 | 5DG3_ITS_Col3 | 5DG4_ITS_Col3 |
| 0.02 pM Std | 0.02 pM Std |
E | 5DG1_16S_Col3 | 5DG2_16S_Col3 | 5DG3_16S_Col3 | 5DG4_16S_Col3 | 5DG5_16S_Col3 | 5DG1_ITS_Col3 | 5DG2_ITS_Col3 | 5DG3_ITS_Col3 | 5DG4_ITS_Col3 |
| 0.2 pM Std | 0.2 pM Std |
F | 5DG1_16S_Col3 | 5DG2_16S_Col3 | 5DG3_16S_Col3 | 5DG4_16S_Col3 | 5DG5_16S_Col3 | 5DG1_ITS_Col3 | 5DG2_ITS_Col3 | 5DG3_ITS_Col3 | 5DG4_ITS_Col3 |
| 2 pM Std | 2 pM Std |
G | 5DG1_16S_Col3 | 5DG2_16S_Col3 | 5DG3_16S_Col3 | 5DG4_16S_Col3 | 5DG5_16S_Col3 | 5DG1_ITS_Col3 | 5DG2_ITS_Col3 | 5DG3_ITS_Col3 | 5DG4_ITS_Col3 |
| 20 pM Std | 20 pM Std |
H | 5DG1_16S_Col3 | 5DG2_16S_Col3 | 5DG3_16S_Col3 | 5DG4_16S_Col3 | 5DG5_16S_Col3 | 5DG1_ITS_Col3 | 5DG2_ITS_Col3 | 5DG3_ITS_Col3 | 5DG4_ITS_Col3 |
|
|
|
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 | 5DG1_16S_Col3 | 5DG2_16S_Col3 | 5DG3_16S_Col3 | 5DG4_16S_Col3 | 5DG5_16S_Col3 | 5DG1_ITS_Col3 | 5DG2_ITS_Col3 | 5DG3_ITS_Col3 | 5DG4_ITS_Col3 | NTC | NTC | NTC |
B | 5DG1_16S_Col3 | 5DG2_16S_Col3 | 5DG3_16S_Col3 | 5DG4_16S_Col3 | 5DG5_16S_Col3 | 5DG1_ITS_Col3 | 5DG2_ITS_Col3 | 5DG3_ITS_Col3 | 5DG4_ITS_Col3 | 0.0002 pM Std | 0.0002 pM Std | 0.0002 pM Std |
C | 5DG1_16S_Col3 | 5DG2_16S_Col3 | 5DG3_16S_Col3 | 5DG4_16S_Col3 | 5DG5_16S_Col3 | 5DG1_ITS_Col3 | 5DG2_ITS_Col3 | 5DG3_ITS_Col3 | 5DG4_ITS_Col3 | 0.002 pM Std | 0.002 pM Std | 0.002 pM Std |
D | 5DG1_16S_Col3 | 5DG2_16S_Col3 | 5DG3_16S_Col3 | 5DG4_16S_Col3 | 5DG5_16S_Col3 | 5DG1_ITS_Col3 | 5DG2_ITS_Col3 | 5DG3_ITS_Col3 | 5DG4_ITS_Col3 | 0.02 pM Std | 0.02 pM Std | 0.02 pM Std |
E | 5DG1_16S_Col3 | 5DG2_16S_Col3 | 5DG3_16S_Col3 | 5DG4_16S_Col3 | 5DG5_16S_Col3 | 5DG1_ITS_Col3 | 5DG2_ITS_Col3 | 5DG3_ITS_Col3 | 5DG4_ITS_Col3 | 0.2 pM Std | 0.2 pM Std | 0.2 pM Std |
F | 5DG1_16S_Col3 | 5DG2_16S_Col3 | 5DG3_16S_Col3 | 5DG4_16S_Col3 | 5DG5_16S_Col3 | 5DG1_ITS_Col3 | 5DG2_ITS_Col3 | 5DG3_ITS_Col3 | 5DG4_ITS_Col3 | 2 pM Std | 2 pM Std | 2 pM Std |
G | 5DG1_16S_Col3 | 5DG2_16S_Col3 | 5DG3_16S_Col3 | 5DG4_16S_Col3 | 5DG5_16S_Col3 | 5DG1_ITS_Col3 | 5DG2_ITS_Col3 | 5DG3_ITS_Col3 | 5DG4_ITS_Col3 | 20 pM Std | 20 pM Std | 20 pM Std |
H | 5DG1_16S_Col3 | 5DG2_16S_Col3 | 5DG3_16S_Col3 | 5DG4_16S_Col3 | 5DG5_16S_Col3 | 5DG1_ITS_Col3 | 5DG2_ITS_Col3 | 5DG3_ITS_Col3 | 5DG4_ITS_Col3 |
|
|
|
Results:
Plate Name | Average Result (nanomoles) |
|---|---|
5DG1_16S_Col3 | 42 |
5DG2_16S_Col3 | 31.72 |
5DG3_16S_Col3 | 76.88 |
5DG4_16S_Col3 | 55.24 |
5DG5_16S_Col3 | 48.91 |
5DG1_ITS_Col3 | 37.74 |
5DG2_ITS_Col3 | 37.41 |
5DG3_ITS_Col3 | 34.67 |
5DG4_ITS_Col3 | 17.29 |
Full qPCR results below:
| View file | ||
|---|---|---|
|
qPCR NovaSeq5_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:
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
A | 5DG5_ITS_Col3 | 5RD1_16S_Col1 | 5RD1_16S_Col2 | 5RD1_16S_Col3 | 5RD1_16S_Col4 | 5RD1_16S_Col5 | 5RD1_16S_Col6 | 5RD1_16S_Col7 | 5RD1_ITS_Col1 |
|
| NTC |
B | 5DG5_ITS_Col3 | 5RD1_16S_Col1 | 5RD1_16S_Col2 | 5RD1_16S_Col3 | 5RD1_16S_Col4 | 5RD1_16S_Col5 | 5RD1_16S_Col6 | 5RD1_16S_Col7 | 5RD1_ITS_Col1 |
| 04.0002 pM Std | 0.0002 pM Std |
C | 5DG5_ITS_Col3 | 5RD1_16S_Col1 | 5RD1_16S_Col2 | 5RD1_16S_Col3 | 5RD1_16S_Col4 | 5RD1_16S_Col5 | 5RD1_16S_Col6 | 5RD1_16S_Col7 | 5RD1_ITS_Col1 |
| 0.002 pM Std | 0.002 pM Std |
D | 5DG5_ITS_Col3 | 5RD1_16S_Col1 | 5RD1_16S_Col2 | 5RD1_16S_Col3 | 5RD1_16S_Col4 | 5RD1_16S_Col5 | 5RD1_16S_Col6 | 5RD1_16S_Col7 | 5RD1_ITS_Col1 |
| 0.02 pM Std | 0.02 pM Std |
E | 5DG5_ITS_Col3 | 5RD1_16S_Col1 | 5RD1_16S_Col2 | 5RD1_16S_Col3 | 5RD1_16S_Col4 | 5RD1_16S_Col5 | 5RD1_16S_Col6 | 5RD1_16S_Col7 | 5RD1_ITS_Col1 |
| 0.2 pM Std | 0.2 pM Std |
F | 5DG5_ITS_Col3 | 5RD1_16S_Col1 | 5RD1_16S_Col2 | 5RD1_16S_Col3 | 5RD1_16S_Col4 | 5RD1_16S_Col5 | 5RD1_16S_Col6 | 5RD1_16S_Col7 | 5RD1_ITS_Col1 |
| 2 pM Std | 2 pM Std |
G | 5DG5_ITS_Col3 | 5RD1_16S_Col1 | 5RD1_16S_Col2 | 5RD1_16S_Col3 | 5RD1_16S_Col4 | 5RD1_16S_Col5 | 5RD1_16S_Col6 | 5RD1_16S_Col7 | 5RD1_ITS_Col1 |
| 20 pM Std | 20 pM Std |
H | 5DG5_ITS_Col3 | 5RD1_16S_Col1 | 5RD1_16S_Col2 | 5RD1_16S_Col3 | 5RD1_16S_Col4 | 5RD1_16S_Col5 | 5RD1_16S_Col6 | 5RD1_16S_Col7 | 5RD1_ITS_Col1 |
|
|
|
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 | 5DG5_ITS_Col3 | 5RD1_16S_Col1 | 5RD1_16S_Col2 | 5RD1_16S_Col3 | 5RD1_16S_Col4 | 5RD1_16S_Col5 | 5RD1_16S_Col6 | 5RD1_16S_Col7 | 5RD1_ITS_Col1 | NTC | NTC | NTC |
B | 5DG5_ITS_Col3 | 5RD1_16S_Col1 | 5RD1_16S_Col2 | 5RD1_16S_Col3 | 5RD1_16S_Col4 | 5RD1_16S_Col5 | 5RD1_16S_Col6 | 5RD1_16S_Col7 | 5RD1_ITS_Col1 | 0.0002 pM Std | 0.0002 pM Std | 0.0002 pM Std |
C | 5DG5_ITS_Col3 | 5RD1_16S_Col1 | 5RD1_16S_Col2 | 5RD1_16S_Col3 | 5RD1_16S_Col4 | 5RD1_16S_Col5 | 5RD1_16S_Col6 | 5RD1_16S_Col7 | 5RD1_ITS_Col1 | 0.002 pM Std | 0.002 pM Std | 0.002 pM Std |
D | 5DG5_ITS_Col3 | 5RD1_16S_Col1 | 5RD1_16S_Col2 | 5RD1_16S_Col3 | 5RD1_16S_Col4 | 5RD1_16S_Col5 | 5RD1_16S_Col6 | 5RD1_16S_Col7 | 5RD1_ITS_Col1 | 0.02 pM Std | 0.02 pM Std | 0.02 pM Std |
E | 5DG5_ITS_Col3 | 5RD1_16S_Col1 | 5RD1_16S_Col2 | 5RD1_16S_Col3 | 5RD1_16S_Col4 | 5RD1_16S_Col5 | 5RD1_16S_Col6 | 5RD1_16S_Col7 | 5RD1_ITS_Col1 | 0.2 pM Std | 0.2 pM Std | 0.2 pM Std |
F | 5DG5_ITS_Col3 | 5RD1_16S_Col1 | 5RD1_16S_Col2 | 5RD1_16S_Col3 | 5RD1_16S_Col4 | 5RD1_16S_Col5 | 5RD1_16S_Col6 | 5RD1_16S_Col7 | 5RD1_ITS_Col1 | 2 pM Std | 2 pM Std | 2 pM Std |
G | 5DG5_ITS_Col3 | 5RD1_16S_Col1 | 5RD1_16S_Col2 | 5RD1_16S_Col3 | 5RD1_16S_Col4 | 5RD1_16S_Col5 | 5RD1_16S_Col6 | 5RD1_16S_Col7 | 5RD1_ITS_Col1 | 20 pM Std | 20 pM Std | 20 pM Std |
H | 5DG5_ITS_Col3 | 5RD1_16S_Col1 | 5RD1_16S_Col2 | 5RD1_16S_Col3 | 5RD1_16S_Col4 | 5RD1_16S_Col5 | 5RD1_16S_Col6 | 5RD1_16S_Col7 | 5RD1_ITS_Col1 |
|
|
|
Results:
Plate Name | Average Result (nanomoles) |
|---|---|
5DG5_ITS_Col3 | 27.22 |
5RD1_16S_Col1 | 2.79 |
5RD1_16S_Col2 | 0.573 |
5RD1_16S_Col3 | 2.64 |
5RD1_16S_Col4 | 8.07 |
5RD1_16S_Col5 | 10.31 |
5RD1_16S_Col6 | 4.6 |
5RD1_16S_Col7 | 13.52 |
5RD1_ITS_Col1 | 5.66 |
Full qPCR results below:
| View file | ||
|---|---|---|
|
qPCR NovaSeq5_Set7
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
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:
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 “NS5 PCR”. (If doing manually, one needs 2 ul of template and 2 ul of the primers).
...
Plate
...
16S Primer
...
ITS Primer
...
5DG3
...
16S0A6
...
ITS0A6
...
16S0B6
...
ITS0B6
...
5DG4
...
16S0C6
...
ITS0C6
...
16S0D6
...
ITS0D6
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_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
11 | 12 | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
A | 5RD1_ITS_Col2 | 5RD1_ITS_Col3 | 5RD1_ITS_Col4 | 5RD1_ITS_Col5 | 5RD1_ITS_Col6 | 5RD1_ITS_Col7 | Ha_Yoon_16S_ITS_Col1 | Ha_Yoon_16S_ITS_Col6 | Ha_Yoon_16S_ITS_Col12 |
|
| NTC |
B | 5RD1_ITS_Col2 | 5RD1_ITS_Col3 | 5RD1_ITS_Col4 | 5RD1_ITS_Col5 | 5RD1_ITS_Col6 | 5RD1_ITS_Col7 | Ha_Yoon_16S_ITS_Col1 | Ha_Yoon_16S_ITS_Col6 | Ha_Yoon_16S_ITS_Col12 |
| 04.0002 pM Std | 0.0002 pM Std |
C | 5RD1_ITS_Col2 | 5RD1_ITS_Col3 | 5RD1_ITS_Col4 | 5RD1_ITS_Col5 | 5RD1_ITS_Col6 | 5RD1_ITS_Col7 | Ha_Yoon_16S_ITS_Col1 | Ha_Yoon_16S_ITS_Col6 | Ha_Yoon_16S_ITS_Col12 |
| 0.002 pM Std | 0.002 pM Std |
D | 5RD1_ITS_Col2 | 5RD1_ITS_Col3 | 5RD1_ITS_Col4 | 5RD1_ITS_Col5 | 5RD1_ITS_Col6 | 5RD1_ITS_Col7 | Ha_Yoon_16S_ITS_Col1 | Ha_Yoon_16S_ITS_Col6 | Ha_Yoon_16S_ITS_Col12 |
| 0.02 pM Std | 0.02 pM Std |
E | 5RD1_ITS_Col2 | 5RD1_ITS_Col3 | 5RD1_ITS_Col4 | 5RD1_ITS_Col5 | 5RD1_ITS_Col6 | 5RD1_ITS_Col7 | Ha_Yoon_16S_ITS_Col1 | Ha_Yoon_16S_ITS_Col6 | Ha_Yoon_16S_ITS_Col12 |
| 0.2 pM Std | 0.2 pM Std |
F | 5RD1_ITS_Col2 | 5RD1_ITS_Col3 | 5RD1_ITS_Col4 | 5RD1_ITS_Col5 | 5RD1_ITS_Col6 | 5RD1_ITS_Col7 | Ha_Yoon_16S_ITS_Col1 | Ha_Yoon_16S_ITS_Col6 | Ha_Yoon_16S_ITS_Col12 |
| 2 pM Std | 2 pM Std |
G | 5RD1_ITS_Col2 | 5RD1_ITS_Col3 | 5RD1_ITS_Col4 | 5RD1_ITS_Col5 | 5RD1_ITS_Col6 | 5RD1_ITS_Col7 | Ha_Yoon_16S_ITS_Col1 | Ha_Yoon_16S_ITS_Col6 | Ha_Yoon_16S_ITS_Col12 |
| 20 pM Std | 20 pM Std |
H | 5RD1_ITS_Col2 | 5RD1_ITS_Col3 | 5RD1_ITS_Col4 | 5RD1_ITS_Col5 | 5RD1_ITS_Col6 | 5RD1_ITS_Col7 | Ha_Yoon_16S_ITS_Col1 | Ha_Yoon_16S_ITS_Col6 | Ha_Yoon_16S_ITS_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:
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
A | 5RD1_ITS_Col2 | 5RD1_ITS_Col3 | 5RD1_ITS_Col4 | 5RD1_ITS_Col5 | 5RD1_ITS_Col6 | 5RD1_ITS_Col7 | Ha_Yoon_16S_ITS_Col1 | Ha_Yoon_16S_ITS_Col6 | Ha_Yoon_16S_ITS_Col12 | NTC | NTC | NTC |
B | 5RD1_ITS_Col2 | 5RD1_ITS_Col3 | 5RD1_ITS_Col4 | 5RD1_ITS_Col5 | 5RD1_ITS_Col6 | 5RD1_ITS_Col7 | Ha_Yoon_16S_ITS_Col1 | Ha_Yoon_16S_ITS_Col6 | Ha_Yoon_16S_ITS_Col12 | 0.0002 pM Std | 0.0002 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
5RD1_ITS_Col2 | 5RD1_ITS_Col3 | 5RD1_ITS_Col4 | 5RD1_ITS_Col5 | 5RD1_ITS_Col6 | 5RD1_ITS_Col7 | Ha_Yoon_16S_ITS_Col1 | Ha_Yoon_16S_ITS_Col6 | Ha_Yoon_16S_ITS_Col12 | 0.002 pM Std | 0.002 pM Std | 0. |
002 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:
...
D | 5RD1_ITS_Col2 | 5RD1_ITS_Col3 | 5RD1_ITS_Col4 | 5RD1_ITS_Col5 | 5RD1_ITS_Col6 | 5RD1_ITS_Col7 | Ha_Yoon_16S_ITS_Col1 | Ha_Yoon_16S_ITS_Col6 | Ha_Yoon_16S_ITS_Col12 | 0.02 pM Std | 0.02 pM Std | 0.02 pM Std |
E | 5RD1_ITS_Col2 | 5RD1_ITS_Col3 | 5RD1_ITS_Col4 | 5RD1_ITS_Col5 | 5RD1_ITS_Col6 | 5RD1_ITS_Col7 | Ha_Yoon_16S_ITS_Col1 | Ha_Yoon_16S_ITS_Col6 | Ha_Yoon_16S_ITS_Col12 | 0.2 pM Std | 0.2 pM Std | 0.2 pM Std |
F | 5RD1_ITS_Col2 | 5RD1_ITS_Col3 | 5RD1_ITS_Col4 | 5RD1_ITS_Col5 | 5RD1_ITS_Col6 | 5RD1_ITS_Col7 | Ha_Yoon_16S_ITS_Col1 | Ha_Yoon_16S_ITS_Col6 | Ha_Yoon_16S_ITS_Col12 | 2 pM Std | 2 pM Std | 2 pM Std |
G | 5RD1_ITS_Col2 | 5RD1_ITS_Col3 | 5RD1_ITS_Col4 | 5RD1_ITS_Col5 | 5RD1_ITS_Col6 | 5RD1_ITS_Col7 | Ha_Yoon_16S_ITS_Col1 | Ha_Yoon_16S_ITS_Col6 | Ha_Yoon_16S_ITS_Col12 | 20 pM Std | 20 pM Std | 20 pM Std |
H | 5RD1_ITS_Col2 | 5RD1_ITS_Col3 | 5RD1_ITS_Col4 | 5RD1_ITS_Col5 | 5RD1_ITS_Col6 | 5RD1_ITS_Col7 | Ha_Yoon_16S_ITS_Col1 | Ha_Yoon_16S_ITS_Col6 | Ha_Yoon_16S_ITS_Col12 |
|
|
|
Results:
Plate Name | Average Result (nanomoles) |
|---|---|
5RD1_ITS_Col2 | 3.89 |
5RD1_ITS_Col3 | 5.49 |
5RD1_ITS_Col4 | 5.19 |
5RD1_ITS_Col5 | 9.37 |
5RD1_ITS_Col6 | 8.81 |
5RD1_ITS_Col7 | 16.43 |
Ha_Yoon_16S_ITS_Col1 | 19.16 |
Ha_Yoon_16S_ITS_Col6 | 18.35 |
Ha_Yoon_16S_ITS_Col12 | 15.73 |
Full qPCR results below:
| View file | ||
|---|---|---|
|