| Info |
|---|
Plates in red have been extracted, PCRed, cleaned, quantified, and normalized by 7-15-22. Bolded red have been qPCRed. |
NS6 Plates
...
6AC1
...
6AC2
...
6AC3
...
6BM1
...
6BM2
...
6LVD1
...
6LVD2
...
6LVD3
...
6LVD4
...
6RD1
...
6RD2
...
6RD3
...
6GTL1
...
6GTL2
...
6RD4
...
6RD5
...
6RD6
...
6RD7
...
6LVD5
...
6LVD6
...
6LVD7
...
MP002
...
MP003
...
MP006
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 label “NS6 PCR”. (If doing manually, one needs 2 ul of template and 2 ul of the primers).
...
Plate
...
16S Primer
...
ITS Primer
...
6AC1
...
16S0A1
...
ITS0A1
...
16S0B1
...
ITS0B1
...
6AC2
...
16S0C1
...
ITS0C1
...
16S0D1
...
ITS0D1
...
6AC3
...
16S0E1
...
ITS0E1
...
16S0F1
...
ITS0F1
...
6BM1
...
16S0G1
...
ITS0G1
...
16S0H1
...
ITS0H1
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)
...
| Info |
|---|
Plates in red have been extracted, PCRed, cleaned, quantified, and normalized by 7-15-22. Bolded red have been qPCRed. |
NS6 Plates
6AC1 | 6AC2 | 6AC3 | |
6BM1 | 6BM2 | ||
6LVD1 | 6LVD2 | 6LVD3 | 6LVD4 |
6RD1 | 6RD2 | 6RD3 | |
6GTL1 | 6GTL2 | ||
6RD4 | 6RD5 | 6RD6 | 6RD7 |
6LVD5 |
|
| |
MP002 | MP003 | MP006 |
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 label “NS6 PCR”. (If doing manually, one needs 2 ul of template and 2 ul of the primers).
Plate | 16S Primer | ITS Primer |
|---|---|---|
6AC1 | 16S0A1 |
ITS0A1 |
16S0B1 |
ITS0B1 |
6AC2 |
16S0C1 |
ITS0C1 |
16S0D1 |
ITS0D1 |
6AC3 |
16S0E1 |
ITS0E1 |
16S0F1 |
ITS0F1 |
6BM1 |
16S0G1 |
ITS0G1 |
16S0H1 |
ITS0H1 |
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)
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 |
|---|
6BM2 |
16S0A2 |
ITS0A2 |
6LVD1 |
16S0C2 |
ITS0C2 |
16S0D2 |
ITS0D2 |
6LVD2 |
16S0E2 |
ITS0E2 |
16S0F2 |
ITS0F2 |
6LVD3 |
16S0G2 |
ITS0G2 |
16S0H2 |
ITS0H2 |
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)
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 |
|---|
6GTL1
16S0G4
ITS0G4
16S0H4
ITS0H4
6GTL2
16S0A5
ITS0A5
16S0B5
6LVD4 | 16S0A3 | ITS0A3 |
16S0B3 | ITS0B3 | |
6RD1 | 16S0C3 | ITS0C3 |
16S0D3 | ITS0D3 | |
6RD2 | 16S0E3 | ITS0E3 |
16S0F3 | ITS0F3 | |
6RD3 | 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”
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 NovaSeq6_Set1
Make 1:1000 dilutions of column 8 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
...
2TRNL1_TRNL
...
2TRNL_16S
...
6AC1_16S
...
6AC1_ITS
...
6AC2_16S
...
6AC2_ITS
...
NTC
...
NTC
...
NTC
...
B
...
2TRNL1_TRNL
...
2TRNL_16S
...
6AC1_16S
...
6AC1_ITS
...
6AC2_16S
...
6AC2_ITS
...
0.0002 pM Std
...
0.0002 pM Std
...
0.0002 pM Std
...
C
...
2TRNL1_TRNL
...
2TRNL_16S
...
6AC1_16S
...
6AC1_ITS
...
6AC2_16S
...
6AC2_ITS
...
0.002 pM Std
...
0.002 pM Std
...
0.002 pM Std
...
D
...
2TRNL1_TRNL
...
2TRNL_16S
...
6AC1_16S
...
6AC1_ITS
...
6AC2_16S
...
6AC2_ITS
...
0.02 pM Std
...
0.02 pM Std
...
0.02 pM Std
...
E
...
2TRNL1_TRNL
...
2TRNL_16S
...
6AC1_16S
...
6AC1_ITS
...
6AC2_16S
...
6AC2_ITS
...
0.2 pM Std
...
0.2 pM Std
...
0.2 pM Std
...
F
...
2TRNL1_TRNL
...
2TRNL_16S
...
6AC1_16S
...
6AC1_ITS
...
6AC2_16S
...
6AC2_ITS
...
2 pM Std
...
2 pM Std
...
2 pM Std
...
G
...
2TRNL1_TRNL
...
2TRNL_16S
...
6AC1_16S
...
6AC1_ITS
...
6AC2_16S
...
6AC2_ITS
...
20 pM Std
...
20 pM Std
...
20 pM Std
...
H
...
2TRNL1_TRNL
...
2TRNL_16S
...
6AC1_16S
...
6AC1_ITS
...
6AC2_16S
...
6AC2_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)
...
110
...
1100
...
2 ul
...
Primer Premix (10X)
...
110
...
220
...
4 ul
...
Ultra Pure Water
...
110
...
440
...
16 ul
...
Total Volume
...
110
...
1760
...
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 |
|---|---|---|
6GTL1 | 16S0G4 | ITS0G4 |
16S0H4 | ITS0H4 | |
6GTL2 | 16S0A5 | ITS0A5 |
16S0B5 | ITS0B5 |
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 NovaSeq6_Set1
Make 1:1000 dilutions of column 8 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 | 2TRNL1_TRNL | 2TRNL_16S | 6AC1_16S | 6AC1_ITS | 6AC2_16S | 6AC2_ITS |
20 pM Std
20 pM Std
NTC | NTC | NTC | |||||||
B | 2TRNL1_TRNL | 2TRNL_16S | 6AC1_16S | 6AC1_ITS | 6AC2_16S | 6AC2_ITS |
0.0002 pM Std |
0.0002 pM Std |
0.0002 pM Std | ||||||||||
C | 2TRNL1_TRNL | 2TRNL_16S | 6AC1_16S | 6AC1_ITS | 6AC2_16S | 6AC2_ITS | 0. |
002 pM Std | 0. |
002 pM Std | 0. |
002 pM Std | ||||||||||||
D | 2TRNL1_TRNL | 2TRNL_16S | 6AC1_16S | 6AC1_ITS | 6AC2_16S | 6AC2_ITS | 0.02 pM Std | 0.02 pM Std | 0.02 pM Std | |||
E | 2TRNL1_TRNL | 2TRNL_16S | 6AC1_16S | 6AC1_ITS | 6AC2_16S | 6AC2_ITS | 0. |
2 pM Std | 0. |
2 pM Std | 0. |
2 pM Std | |||||||||
F | 2TRNL1_TRNL | 2TRNL_16S | 6AC1_16S | 6AC1_ITS | 6AC2_16S | 6AC2_ITS |
2 pM Std |
2 pM Std |
2 pM Std | |||||||||
G | 2TRNL1_TRNL | 2TRNL_16S | 6AC1_16S | 6AC1_ITS | 6AC2_16S | 6AC2_ITS |
NTC
NTC
20 pM Std | 20 pM Std | 20 pM Std | |||||||
H | 2TRNL1_TRNL | 2TRNL_16S | 6AC1_16S | 6AC1_ITS | 6AC2_16S | 6AC2_ITS |
|
|
NTC
Results:
...
Plate Name
...
Average Result (nanomoles)
...
2TRNL1_TRNL
...
10.63
...
2TRNL_16S
...
6.02E-01
...
6AC1_16S
...
23.35
...
6AC1_ITS
...
36.97
...
6AC2_16S
...
67.91
...
6AC2_ITS
...
28.37
Full qPCR results below:
| View file | ||
|---|---|---|
|
qPCR NovaSeq6_Set2
...
|
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 |
2TRNL1_ |
TRNL |
2TRNL_16S |
6AC1_ |
16S |
6AC1_ITS |
6AC2_16S |
6AC2_ITS |
6LVD1_16S
6GTL1_ITS
20 pM Std | 20 pM Std | 20 pM Std |
B |
6AC3_ITS
6AC3_16S
6RD1_ITS
2TRNL1_TRNL | 2TRNL_16S | 6AC1_16S |
6AC1_ITS |
6AC2_16S |
6AC2_ITS |
6LVD1_16S
6GTL1_ITS
2 pM Std | 2 pM Std | 2 pM Std |
C |
2TRNL1_ |
TRNL |
2TRNL_16S |
6RD1_ITS
6AC1_16S |
6AC1_ITS |
6AC2_16S |
6AC2_ITS |
6LVD1_16S
6GTL1_ITS
0.2 pM Std | 0.2 pM Std | 0.2 pM Std |
D |
6AC3_ITS
6AC3_16S
6RD1_ITS
2TRNL1_TRNL | 2TRNL_16S |
6AC1_ |
16S |
6AC1_ITS |
6AC2_16S |
6AC2_ITS |
0.02 pM Std | 0.02 pM Std | 0.02 pM Std | |
E |
2TRNL1_ |
TRNL |
2TRNL_16S |
6RD1_ITS
6AC1_16S |
6AC1_ITS |
6AC2_16S |
6AC2_ITS |
6LVD1_16S
6GTL1_ITS
0.002 pM Std | 0.002 pM Std | 0.002 pM Std |
F |
6AC3_ITS
6AC3_16S
6RD1_ITS
2TRNL1_TRNL | 2TRNL_16S | 6AC1_16S |
6AC1_ITS |
6AC2_16S |
6AC2_ITS |
6LVD1_16S
6GTL1_ITS
0.0002 pM Std | 0.0002 pM Std | 0.0002 pM Std |
G |
2TRNL1_ |
TRNL |
2TRNL_16S |
6RD1_ITS
6AC1_16S |
6AC1_ITS |
6AC2_16S |
6AC2_ITS |
6LVD1_16S
6GTL1_ITS
NTC | NTC | NTC |
H |
2TRNL1_ |
TRNL |
2TRNL_16S |
6RD1_ITS
6RD1_16S
6BM1_ITS
6AC1_16S |
6AC1_ITS |
6AC2_16S |
6AC2_ITS |
6GTL1_16S
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
...
NTC | NTC | NTC |
Results:
Plate Name | Average Result (nanomoles) |
|---|---|
2TRNL1_TRNL | 10.63 |
2TRNL_16S | 6.02E-01 |
6AC1_16S | 23.35 |
6AC1_ITS | 36.97 |
6AC2_16S | 67.91 |
6AC2_ITS | 28.37 |
Full qPCR results below:
| View file | ||
|---|---|---|
|
qPCR NovaSeq6_Set2
Make 1:1000 dilutions of column 8 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 | 6AC3_ITS | 6AC3_16S | 6RD1_ITS | 6RD1_16S | 6BM1_ITS | 6BM1_16S | 6LVD1_ITS | 6LVD1_16S | 6GTL1_ITS | 6GTL1_16S |
20 pM Std
20 pM Std
B | 6AC3_ITS | 6AC3_16S | 6RD1_ITS | 6RD1_16S | 6BM1_ITS | 6BM1_16S | 6LVD1_ITS | 6LVD1_16S | 6GTL1_ITS | 6GTL1_16S |
2 pM Std
C |
6AC3_ITS
6AC3_16S
6RD1_ITS
6RD1_16S
6BM1_ITS
6BM1_16S
6LVD1_ITS
6LVD1_16S
6GTL1_ITS
6GTL1_16S
D
0.2 pM Std
6AC3_ITS | 6AC3_16S | 6RD1_ITS | 6RD1_16S | 6BM1_ITS | 6BM1_16S | 6LVD1_ITS | 6LVD1_16S | 6GTL1_ITS |
6GTL1_16S
0.02 pM Std
0.02 pM Std
6GTL1_16S | ||||||||||
D | 6AC3_ITS | 6AC3_16S | 6RD1_ITS | 6RD1_16S | 6BM1_ITS | 6BM1_16S | 6LVD1_ITS | 6LVD1_16S | 6GTL1_ITS | 6GTL1_16S |
0.002 pM Std
0.002 pM Std
G
E | 6AC3_ITS | 6AC3_16S | 6RD1_ITS | 6RD1_16S | 6BM1_ITS | 6BM1_16S | 6LVD1_ITS | 6LVD1_16S | 6GTL1_ITS | 6GTL1_16S |
0.0002 pM Std
0.0002 pM Std
F | 6AC3_ITS | 6AC3_16S | 6RD1_ITS | 6RD1_16S | 6BM1_ITS | 6BM1_16S | 6LVD1_ITS | 6LVD1_16S | 6GTL1_ITS | 6GTL1_16S |
NTC
NTC
G | 6AC3_ITS | 6AC3_16S | 6RD1_ITS | 6RD1_16S | 6BM1_ITS | 6BM1_16S | 6LVD1_ITS | 6LVD1_16S | 6GTL1_ITS | 6GTL1_16S |
NTC
NTC
Results:
Plate Name
Average Result (nanomoles)
H | 6AC3_ITS |
6AC3_16S |
6RD1_ITS |
10.152
6RD1_16S |
15.709
6BM1_ITS |
6BM1_ |
16S | 6LVD1_ITS |
4.532
6LVD1_16S |
8.102
6GTL1_ITS |
5.992
6GTL1_16S |
Needs to be redone
Full qPCR results below:
| View file | ||
|---|---|---|
|
qPCR NovaSeq6_Set3
Make 1:1000 dilutions of column 8 from the PCR plates by adding 1 ul to 999 ul TE in a deep well plateAdd 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 |
6AC3_ITS |
6AC3_16S |
6RD1_ |
ITS |
6RD1_ |
16S |
6BM1_ |
ITS |
6BM1_16S |
6LVD1_ |
ITS |
6LVD1_ |
16S | 6GTL1_ |
ITS |
6GTL1_16S |
20 pM Std | 20 pM Std |
B |
6AC3_ITS |
6AC3_16S | 6RD1_ITS |
6RD1_16S |
6BM1_ |
ITS |
6BM1_ |
16S |
6LVD1_ |
ITS |
6LVD1_16S |
6GTL1_ITS | 6GTL1_16S |
2 |
pM Std |
2 pM Std |
C |
6AC3_ITS |
6AC3_16S | 6RD1_ITS |
6RD1_16S |
6BM1_ |
ITS |
6BM1_ |
16S |
6LVD1_ |
ITS |
6LVD1_16S |
6GTL1_ITS | 6GTL1 |
_16S |
0. |
2 pM Std | 0. |
2 pM Std |
D |
6AC3_ITS |
6AC3_16S |
6RD1_ITS | 6RD1_16S |
6BM1_ |
ITS |
6BM1_ |
16S |
6LVD1_ |
ITS |
6LVD1_16S |
6GTL1_ITS | 6GTL1_16S |
2TRNL1_16S_2
0.02 pM Std | 0.02 pM Std |
E |
6AC3_ITS |
6AC3_16S | 6RD1_ITS |
6RD1_16S |
6BM1_ |
ITS |
6BM1_ |
16S |
6LVD1_ |
ITS |
6LVD1_16S |
6GTL1_ITS | 6GTL1 |
_16S |
0. |
002 pM Std | 0. |
002 pM Std |
F |
6AC3_ITS |
6AC3_16S |
6RD1_ |
ITS |
6RD1_ |
16S |
6BM1_ |
ITS |
6BM1_16S |
6LVD1_ITS | 6LVD1_16S |
6GTL1_ITS | 6GTL1_16S |
2TRNL1_16S_2
0.0002 pM Std |
0.0002 pM Std |
G |
6AC3_ITS |
6AC3_16S |
6RD1_ITS | 6RD1_16S |
6BM1_ |
ITS |
6BM1_ |
16S |
6LVD1_ |
ITS |
6LVD1_16S |
6GTL1_ITS | 6GTL1_16S |
2TRNL1_16S_2
20 pM Std
NTC | NTC |
H |
6AC3_ITS |
6AC3_16S |
6RD1_ITS | 6RD1_16S |
6BM1_ |
ITS |
6BM1_ |
16S |
6LVD1_ |
ITS |
6LVD1_16S |
6GTL1_ITS | 6GTL1_16S |
2TRNL1_16S_2
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
...
NTC | NTC |
Results:
Plate Name | Average Result (nanomoles) |
|---|---|
6AC3_ITS | 4.125 |
6AC3_16S | 7.46 |
6RD1_ITS | 10.152 |
6RD1_16S | 15.709 |
6BM1_ITS | 7.874 |
6BM1_16S | 4.842 |
6LVD1_ITS | 4.532 |
6LVD1_16S | 8.102 |
6GTL1_ITS | 5.992 |
6GTL1_16S | Needs to be redone |
Full qPCR results below:
| View file | ||
|---|---|---|
|
qPCR NovaSeq6_Set3
Make 1:1000 dilutions of column 8 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
6LVD2_ITS
6LVD2_16S
2TRNL3_16S
2TRNL3_TRNL
2TRNL2_TRNL
2TRNL2_16S
6RD2_16S
6RD2_ITS
6GTL1_16S
2TRNL1_16S_2
20 pM Std
20 pM Std
12 | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|
A | 6LVD2_ITS | 6LVD2_16S | 2TRNL3_16S | 2TRNL3_TRNL | 2TRNL2_TRNL | 2TRNL2_16S | 6RD2_16S | 6RD2_ITS | 6GTL1_16S | 2TRNL1_16S_2 |
2 pM Std
2 pM Std
| NTC | |||||||||
B | 6LVD2_ITS | 6LVD2_16S | 2TRNL3_16S | 2TRNL3_TRNL | 2TRNL2_TRNL | 2TRNL2_16S | 6RD2_16S | 6RD2_ITS | 6GTL1_16S | 2TRNL1_16S_2 |
04. |
0002 pM Std | 0. |
0002 pM Std |
C | 6LVD2_ITS | 6LVD2_16S | 2TRNL3_16S | 2TRNL3_TRNL | 2TRNL2_TRNL | 2TRNL2_16S | 6RD2_16S | 6RD2_ITS | 6GTL1_16S | 2TRNL1_16S_2 | 0. |
002 pM Std | 0. |
002 pM Std |
D | 6LVD2_ITS | 6LVD2_16S | 2TRNL3_16S | 2TRNL3_TRNL | 2TRNL2_TRNL | 2TRNL2_16S | 6RD2_16S | 6RD2_ITS | 6GTL1_16S | 2TRNL1_16S_2 | 0. |
02 pM Std | 0. |
02 pM Std |
E | 6LVD2_ITS | 6LVD2_16S | 2TRNL3_16S | 2TRNL3_TRNL | 2TRNL2_TRNL | 2TRNL2_16S | 6RD2_16S | 6RD2_ITS | 6GTL1_16S | 2TRNL1_16S_2 | 0. |
2 pM Std | 0. |
2 pM Std |
F | 6LVD2_ITS | 6LVD2_16S | 2TRNL3_16S | 2TRNL3_TRNL | 2TRNL2_TRNL | 2TRNL2_16S | 6RD2_16S | 6RD2_ITS | 6GTL1_16S | 2TRNL1_16S_2 |
NTC
NTC
2 pM Std | 2 pM Std | |||||||||
G | 6LVD2_ITS | 6LVD2_16S | 2TRNL3_16S | 2TRNL3_TRNL | 2TRNL2_TRNL | 2TRNL2_16S | 6RD2_16S | 6RD2_ITS | 6GTL1_16S | 2TRNL1_16S_2 |
NTC
NTC
Results:
Plate Name
Average Result (nanomoles)
20 pM Std | 20 pM Std |
H | 6LVD2_ITS |
16.09
6LVD2_16S |
2TRNL3_16S |
2TRNL3_TRNL |
2TRNL2_TRNL |
7.51
2TRNL2_16S |
36.27
6RD2_16S |
6RD2_ITS |
29.45
6GTL1_16S
8.2
2TRNL1_16S_2
32.72
Full qPCR results below:
| View file | ||
|---|---|---|
|
qPCR NovaSeq6_Set4
...
6GTL1_16S | 2TRNL1_16S_2 |
|
|
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:
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
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
A |
NTC
6LVD2_ITS | 6LVD2_16S | 2TRNL3_16S | 2TRNL3_TRNL | 2TRNL2_TRNL | 2TRNL2_16S | 6RD2_16S | 6RD2_ITS | 6GTL1_16S | 2TRNL1_16S_2 | 20 pM Std | 20 pM Std |
Add 4 ul of template, pool, or standards to each well:
...
...
1
...
2
...
3
...
4
...
5
...
6
...
7
...
8
...
9
...
10
...
11
...
12
...
A
...
6RD4_16S
...
6RD4_ITS
...
6RD5_16S
...
6RD5_ITS
...
6RD6_16S
...
6RD6_ITS
...
6RD7_16S
...
6RD7_ITS
...
6LVD5_16S
...
NTC
...
NTC
...
NTC
...
B
...
6RD4_16S
...
6RD4_ITS
...
6RD5_16S
...
6RD5_ITS
...
6RD6_16S
...
6RD6_ITS
...
6RD7_16S
...
6RD7_ITS
...
6LVD5_16S
...
0.0002 pM Std
...
0.0002 pM Std
...
0.0002 pM Std
...
C
...
6RD4_16S
...
6RD4_ITS
...
6RD5_16S
...
6RD5_ITS
...
6RD6_16S
...
6RD6_ITS
...
6RD7_16S
...
6RD7_ITS
...
6LVD5_16S
...
0.002 pM Std
...
0.002 pM Std
...
0.002 pM Std
...
D
...
6RD4_16S
...
6RD4_ITS
...
6RD5_16S
...
6RD5_ITS
...
6RD6_16S
...
6RD6_ITS
...
6RD7_16S
...
6RD7_ITS
...
6LVD5_16S
...
0.02 pM Std
...
0.02 pM Std
...
0.02 pM Std
...
E
...
6RD4_16S
...
6RD4_ITS
...
6RD5_16S
...
6RD5_ITS
...
6RD6_16S
...
6RD6_ITS
...
6RD7_16S
...
6RD7_ITS
...
6LVD5_ITS
...
0.2 pM Std
...
0.2 pM Std
...
0.2 pM Std
...
F
...
6RD4_16S
...
6RD4_ITS
...
6RD5_16S
...
6RD5_ITS
...
6RD6_16S
...
6RD6_ITS
...
6RD7_16S
...
6RD7_ITS
...
6LVD5_ITS
...
2 pM Std
...
2 pM Std
...
2 pM Std
...
G
...
6RD4_16S
...
6RD4_ITS
...
6RD5_16S
...
6RD5_ITS
...
6RD6_16S
...
6RD6_ITS
...
6RD7_16S
...
6RD7_ITS
...
6LVD5_ITS
...
20 pM Std
...
20 pM Std
...
20 pM Std
...
H
...
6RD4_16S
...
6RD4_ITS
...
6RD5_16S
...
6RD5_ITS
...
6RD6_16S
...
6RD6_ITS
...
6RD7_16S
...
6RD7_ITS
...
6LVD5_ITS
...
...
...
Results:
...
Plate Name
...
Average Result (nanomoles)
...
6RD4_16S
...
6RD4_ITS
...
6RD5_16S
...
6RD5_ITS
...
6RD6_16S
...
6RD6_ITS
...
6RD7_16S
...
6RD7_ITS
...
6LVD5_16S_ITS
Full qPCR results below:
qPCR NovaSeq6_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
NTC
B
04.0002 pM Std
0.0002 pM Std
C
0.002 pM Std
0.002 pM Std
B |
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
6LVD2_ITS | 6LVD2_16S | 2TRNL3_16S | 2TRNL3_TRNL | 2TRNL2_TRNL | 2TRNL2_16S | 6RD2_16S | 6RD2_ITS | 6GTL1_16S | 2TRNL1_16S_2 | 2 pM Std | 2 pM Std | |
C | 6LVD2_ITS | 6LVD2_16S | 2TRNL3_16S | 2TRNL3_TRNL | 2TRNL2_TRNL | 2TRNL2_16S | 6RD2_16S | 6RD2_ITS | 6GTL1_16S | 2TRNL1_16S_2 | 0.2 pM Std | 0.2 pM Std |
D | 6LVD2_ITS | 6LVD2_16S | 2TRNL3_16S | 2TRNL3_TRNL | 2TRNL2_TRNL | 2TRNL2_16S | 6RD2_16S | 6RD2_ITS | 6GTL1_16S | 2TRNL1_16S_2 | 0.02 pM Std | 0.02 pM Std |
E | 6LVD2_ITS | 6LVD2_16S | 2TRNL3_16S | 2TRNL3_TRNL | 2TRNL2_TRNL | 2TRNL2_16S | 6RD2_16S | 6RD2_ITS | 6GTL1_16S | 2TRNL1_16S_2 | 0.002 pM Std | 0.002 pM Std |
F | 6LVD2_ITS | 6LVD2_16S | 2TRNL3_16S | 2TRNL3_TRNL | 2TRNL2_TRNL | 2TRNL2_16S | 6RD2_16S | 6RD2_ITS | 6GTL1_16S | 2TRNL1_16S_2 | 0.0002 pM Std | 0.0002 pM Std |
G | 6LVD2_ITS | 6LVD2_16S | 2TRNL3_16S | 2TRNL3_TRNL | 2TRNL2_TRNL | 2TRNL2_16S | 6RD2_16S | 6RD2_ITS | 6GTL1_16S | 2TRNL1_16S_2 | NTC | NTC |
H | 6LVD2_ITS | 6LVD2_16S | 2TRNL3_16S | 2TRNL3_TRNL | 2TRNL2_TRNL | 2TRNL2_16S | 6RD2_16S | 6RD2_ITS | 6GTL1_16S | 2TRNL1_16S_2 | NTC | NTC |
Results:
Plate Name | Average Result (nanomoles) |
|---|---|
6LVD2_ITS | 16.09 |
6LVD2_16S | 21.71 |
2TRNL3_16S | 42.69 |
2TRNL3_TRNL | 8.68 |
2TRNL2_TRNL | 7.51 |
2TRNL2_16S | 36.27 |
6RD2_16S | 28.48 |
6RD2_ITS | 29.45 |
6GTL1_16S | 8.2 |
2TRNL1_16S_2 | 32.72 |
Full qPCR results below:
| View file | ||
|---|---|---|
|
qPCR NovaSeq6_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 | 6RD4_16S_Col3 | 6RD4_ITS_Col3 | 6RD5_16S_Col3 | 6RD5_ITS_Col3 | 6RD6_16S_Col3 | 6RD6_ITS_Col3 | 6RD7_16S_Col3 | 6RD7_ITS_Col3 | 6LVD5_16S_A1 |
|
| NTC |
B | 6RD4_16S_Col3 | 6RD4_ITS_Col3 | 6RD5_16S_Col3 | 6RD5_ITS_Col3 | 6RD6_16S_Col3 | 6RD6_ITS_Col3 | 6RD7_16S_Col3 | 6RD7_ITS_Col3 | 6LVD5_16S_B1 |
| 04.0002 pM Std | 0.0002 pM Std |
C | 6RD4_16S_Col3 | 6RD4_ITS_Col3 | 6RD5_16S_Col3 | 6RD5_ITS_Col3 | 6RD6_16S_Col3 | 6RD6_ITS_Col3 | 6RD7_16S_Col3 | 6RD7_ITS_Col3 | 6LVD5_16S_C1 |
| 0.002 pM Std | 0.002 pM Std |
D | 6RD4_16S_Col3 | 6RD4_ITS_Col3 | 6RD5_16S_Col3 | 6RD5_ITS_Col3 | 6RD6_16S_Col3 | 6RD6_ITS_Col3 | 6RD7_16S_Col3 | 6RD7_ITS_Col3 | 6LVD5_16S_D1 |
| 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)
...
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:
...
Plate Name
...
Average Result (nanomoles)
Full qPCR results below:
qPCR NovaSeq6_Set6
...
6RD4_16S_Col3 | 6RD4_ITS_Col3 | 6RD5_16S_Col3 | 6RD5_ITS_Col3 | 6RD6_16S_Col3 | 6RD6_ITS_Col3 | 6RD7_16S_Col3 | 6RD7_ITS_Col3 | 6LVD5_ITS_A8 |
| 0.2 pM Std | 0.2 pM Std | |
F | 6RD4_16S_Col3 | 6RD4_ITS_Col3 | 6RD5_16S_Col3 | 6RD5_ITS_Col3 | 6RD6_16S_Col3 | 6RD6_ITS_Col3 | 6RD7_16S_Col3 | 6RD7_ITS_Col3 | 6LVD5_ITS_B8 |
| 2 pM Std | 2 pM Std |
G | 6RD4_16S_Col3 | 6RD4_ITS_Col3 | 6RD5_16S_Col3 | 6RD5_ITS_Col3 | 6RD6_16S_Col3 | 6RD6_ITS_Col3 | 6RD7_16S_Col3 | 6RD7_ITS_Col3 | 6LVD5_ITS_C8 |
| 20 pM Std | 20 pM Std |
H | 6RD4_16S_Col3 | 6RD4_ITS_Col3 | 6RD5_16S_Col3 | 6RD5_ITS_Col3 | 6RD6_16S_Col3 | 6RD6_ITS_Col3 | 6RD7_16S_Col3 | 6RD7_ITS_Col3 | 6LVD5_ITS_D8 |
|
|
|
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:
...
G
...
20 pM Std
...
20 pM Std
...
20 pM Std
...
H
...
...
...
Results:
...
Plate Name
...
Average Result (nanomoles)
...
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
|---|
9
10
11
12
A
NTC
B
9 | 10 | 11 | 12 | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
A | 6RD4_16S | 6RD4_ITS | 6RD5_16S | 6RD5_ITS | 6RD6_16S | 6RD6_ITS | 6RD7_16S | 6RD7_ITS | 6LVD5_16S | NTC | NTC | NTC |
B | 6RD4_16S | 6RD4_ITS | 6RD5_16S | 6RD5_ITS | 6RD6_16S | 6RD6_ITS | 6RD7_16S | 6RD7_ITS | 6LVD5_16S | 0.0002 pM Std | 0.0002 pM Std | 0.0002 pM Std |
C |
6RD4_16S | 6RD4_ITS | 6RD5_16S | 6RD5_ITS | 6RD6_16S | 6RD6_ITS | 6RD7_16S | 6RD7_ITS | 6LVD5_16S | 0.002 pM Std | 0.002 pM Std | 0.002 pM Std | |
D |
6RD4_16S | 6RD4_ITS | 6RD5_16S | 6RD5_ITS | 6RD6_16S | 6RD6_ITS | 6RD7_16S | 6RD7_ITS | 6LVD5_16S | 0.02 pM Std | 0.02 pM Std | 0.02 pM Std | |
E |
6RD4_16S | 6RD4_ITS | 6RD5_16S | 6RD5_ITS | 6RD6_16S | 6RD6_ITS | 6RD7_16S | 6RD7_ITS | 6LVD5_ITS | 0.2 pM Std |
F
0.2 pM Std | 0.2 pM Std |
F |
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
6RD4_16S | 6RD4_ITS | 6RD5_16S | 6RD5_ITS | 6RD6_16S | 6RD6_ITS | 6RD7_16S | 6RD7_ITS | 6LVD5_ITS | 2 pM Std | 2 pM Std | 2 pM Std | |
G | 6RD4_16S | 6RD4_ITS | 6RD5_16S | 6RD5_ITS | 6RD6_16S | 6RD6_ITS | 6RD7_16S | 6RD7_ITS | 6LVD5_ITS | 20 pM Std | 20 pM Std | 20 pM Std |
H | 6RD4_16S | 6RD4_ITS | 6RD5_16S | 6RD5_ITS | 6RD6_16S | 6RD6_ITS | 6RD7_16S | 6RD7_ITS | 6LVD5_ITS | NS6_Pool_1:1000 | NS6_Pool_1:1000 | NS6_Pool_1:1000 |
Results:
Plate Name | Average Result (nanomoles) |
|---|---|
6RD4_16S | 70.66 |
6RD4_ITS | 22.2 |
6RD5_16S | 76.68 |
6RD5_ITS | 86.82 |
6RD6_16S | 32.57 |
6RD6_ITS | 45.66 |
6RD7_16S | 45.71 |
6RD7_ITS | 67.98 |
6LVD5_16S | 50.03 |
6LVD5_ITS | 14.98 |
NS6_Pool | 12.17 |
Full qPCR results below:
| View file | ||
|---|---|---|
|