*Make Protocol Below for 2017 GBS Plates….
Prepare two ITS and two 16S PCR plates for the following template plates:
ALF_PLT1_2020_END | ALF_PLT2_2020_END | ALF_PLT3_2020_END | ALF_PLT4_2020_END |
ALF_PLT5_2020_END | ALF_PLT6_2020_END | ALF_PLT7_2020_END | ALF_PLT8_2020_END |
ALF_PLT1_2020_EPI | ALF_PLT2_2020_EPI | ALF_PLT3_2020_EPI | ALF_PLT4_2020_EPI |
ALF_PLT5_2020_EPI | ALF_PLT6_2020_EPI | ALF_PLT7_2020_EPI | ALF_PLT8_2020_EPI |
ALF_PLT9_2020_EPI | ALF_PLT1_2020_SOIL | ||
ALF_PLT1_2017_GBS | ALF_PLT2_2017_GBS | ALF_PLT3_2017_GBS | ALF_PLT4_2017_GBS |
ALF_PLT5_2017_GBS | ALF_PLT6_2017_GBS | ALF_PLT7_2017_GBS | ALF_PLT8_2017_GBS |
ALF_PLT9_2017_GBS | ALF_PLT10_2017_GBS | ALF_PLT11_2017_GBS |
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 | Ultra Pure H2O | 1700 |
12,325 | ||
11 | Total Volume | 1700 |
18,700 |
Add 11 ul to each well of a hard shell, full skirt plate. Seal with bubble strips and store in refrigerator until needed label “ALF PCR”. (If doing manually, one needs 2 ul of template and 2 ul of the primers).
Plates | 16S MIDs | ITS MIDs |
|---|---|---|
ALF_PLT1_2020_END | long16S0A1 | longITS0A1 |
long16S0B1 | longITS0B1 | |
ALF_PLT2_2020_END | long16S0C1 | longITS0C1 |
long16S0D1 | longITS0D1 | |
ALF_PLT3_2020_END | long16S0E1 | longITS0E1 |
long16S0F1 | longITS0F1 | |
ALF_PLT4_2020_END | long16S0G1 | longITS0G1 |
long16S0H1 | longITS0H1 |
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 ALF_END_RUN1
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
...
ALF_END_PLT1_16S
...
ALF_END_PLT1_ITS
...
ALF_END_PLT2_16S
...
ALF_END_PLT2_ITS
...
ALF_END_PLT3_16S
...
ALF_END_PLT3_ITS
...
ALF_END_PLT4_16S
...
ALF_END_PLT4_ITS
...
...
NTC
...
NTC
...
NTC
...
B
...
ALF_END_PLT1_16S
...
ALF_END_PLT1_ITS
...
ALF_END_PLT2_16S
...
ALF_END_PLT2_ITS
...
ALF_END_PLT3_16S
...
ALF_END_PLT3_ITS
...
ALF_END_PLT4_16S
...
ALF_END_PLT4_ITS
...
...
0.0002 pM Std
...
0.0002 pM Std
...
0.0002 pM Std
...
C
...
ALF_END_PLT1_16S
...
ALF_END_PLT1_ITS
...
ALF_END_PLT2_16S
...
ALF_END_PLT2_ITS
...
ALF_END_PLT3_16S
...
ALF_END_PLT3_ITS
...
ALF_END_PLT4_16S
...
ALF_END_PLT4_ITS
...
...
0.002 pM Std
...
0.002 pM Std
...
0.002 pM Std
...
D
...
ALF_END_PLT1_16S
...
ALF_END_PLT1_ITS
...
ALF_END_PLT2_16S
...
ALF_END_PLT2_ITS
...
ALF_END_PLT3_16S
...
ALF_END_PLT3_ITS
...
ALF_END_PLT4_16S
...
ALF_END_PLT4_ITS
...
...
0.02 pM Std
...
0.02 pM Std
...
0.02 pM Std
...
E
...
ALF_END_PLT1_16S
...
ALF_END_PLT1_ITS
...
ALF_END_PLT2_16S
...
ALF_END_PLT2_ITS
...
ALF_END_PLT3_16S
...
ALF_END_PLT3_ITS
...
ALF_END_PLT4_16S
...
ALF_END_PLT4_ITS
...
...
0.2 pM Std
...
0.2 pM Std
...
0.2 pM Std
...
F
...
ALF_END_PLT1_16S
...
ALF_END_PLT1_ITS
...
ALF_END_PLT2_16S
...
ALF_END_PLT2_ITS
...
ALF_END_PLT3_16S
...
ALF_END_PLT3_ITS
...
ALF_END_PLT4_16S
...
ALF_END_PLT4_ITS
...
...
2 pM Std
...
2 pM Std
...
2 pM Std
...
G
...
ALF_END_PLT1_16S
...
ALF_END_PLT1_ITS
...
ALF_END_PLT2_16S
...
ALF_END_PLT2_ITS
...
ALF_END_PLT3_16S
...
ALF_END_PLT3_ITS
...
ALF_END_PLT4_16S
...
ALF_END_PLT4_ITS
...
...
20 pM Std
...
20 pM Std
...
20 pM Std
...
H
...
ALF_END_PLT1_16S
...
ALF_END_PLT1_ITS
...
ALF_END_PLT2_16S
...
ALF_END_PLT2_ITS
...
ALF_END_PLT3_16S
...
ALF_END_PLT3_ITS
...
ALF_END_PLT4_16S
...
ALF_END_PLT4_ITS
...
...
...
...
...
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 | Ultra Pure H2O | 1700 | 12,325 |
11 | Total Volume | 1700 | 18,700 |
Add 11 ul to each well of a hard shell, full skirt plate. Seal with bubble strips and store in refrigerator until needed label “ALF PCR”. (If doing manually, one needs 2 ul of template and 2 ul of the primers).
Plates | 16S MIDs | ITS MIDs |
|---|---|---|
ALF_PLT5_2020_END | long16S0A2 | longITS0A2 |
long16S0B2 | longITS0B2 | |
ALF_PLT6_2020_END | long16S0C2 | longITS0C2 |
long16S0D2 | longITS0D2 | |
ALF_PLT7_2020_END | long16S0E2 | longITS0E2 |
long16S0F2 | longITS0F2 | |
ALF_PLT8_2020_END | long16S0G2 | longITS0G2 |
long16S0H2 | longITS0H2 |
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 ALF_END
Make 1:1000 dilutions 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 | ALF_END_PLT1_16S_3B | ALF_END_PLT3_16S_3B | ALF_END_PLT5_16S_3B | ALF_END_PLT7_16S_3B |
|
|
| NTC | ||||
B | ALF_END_PLT1_16S_9H | ALF_END_PLT3_16S_9H | ALF_END_PLT5_16S_9H | ALF_END_PLT7_16S_9H |
|
| 04.0002 pM Std | 0.0002 pM Std | ||||
C | ALF_END_PLT1_ITS_3B | ALF_END_PLT5_ITS_3B | ALF_END_PLT3_ITS_3B | ALF_END_PLT7_ITS_3B |
|
| 0.002 pM Std | 0.002 pM Std | ||||
D | ALF_END_PLT1_ITS_9H | ALF_END_PLT5_ITS_9H | ALF_END_PLT3_ITS_9H | ALF_END_PLT7_ITS_9H |
|
| 0.02 pM Std | 0.02 pM Std | ||||
E | ALF_END_PLT2_16S_3B | ALF_END_PLT4_16S_3B | ALF_END_PLT6_16S_3B | ALF_END_PLT8_16S_3B |
|
| 0.2 pM Std | 0.2 pM Std | ||||
F | ALF_END_PLT2_16S_9H | ALF_END_PLT4_16S_9H | ALF_END_PLT6_16S_9H | ALF_END_PLT8_16S_9H |
|
| 2 pM Std | 2 pM Std | ||||
G | ALF_END_PLT2_ITS_3B | ALF_END_PLT4_ITS_3B | ALF_END_PLT6_ITS_3B | ALF_END_PLT8_ITS_3B |
|
| 20 pM Std | 20 pM Std | ||||
H | ALF_END_PLT2_ITS_9H | ALF_END_PLT4_ITS_9H | ALF_END_PLT6_ITS_9H | ALF_END_PLT8_ITS_9H |
Add 16 ul of Illumina Library Quantification MasterMix to each well:
ul/rxn | Reagent | # of rxns | ul needed |
|---|---|---|---|
10 ul | KAPA SYBR FAST qPCR MM (2X) |
60 |
600 | |
2 ul | Primer Premix (10X) |
60 |
120 | |
4 ul | Ultra Pure Water |
60 |
240 | |
16 ul | Total Volume |
60 |
960 |
Add 4 ul of template, pool, or standards to each well:
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
A | ALF_END_PLT1_16S_3B | ALF_END_PLT3_ |
16S_ |
3B | ALF_END_ |
PLT5_16S_3B | ALF_END_PLT7_ |
16S_3B |
| NTC | NTC | NTC | ||||
B | ALF_END_ |
PLT1_16S_9H | ALF_END_PLT3_ |
16S_9H | ALF_END_ |
PLT5_16S_9H | ALF_END_PLT7_ |
16S_ |
9H |
|
NTC
NTC
NTC
B
0.0002 pM Std | 0.0002 pM Std | 0.0002 pM Std |
C | ALF_END_PLT1_ITS |
_3B | ALF_END_ |
PLT5_ITS |
_3B | ALF_END_PLT3_ITS |
_3B | ALF_END_ |
PLT7_ITS_3B |
| 0. |
002 pM Std | 0. |
002 pM Std | 0. |
002 pM Std |
D | ALF_END_PLT1_ITS_ |
9H | ALF_END_ |
PLT5_ITS_9H | ALF_END_ |
PLT3_ |
ITS_9H | ALF_END_ |
PLT7_ITS_9H |
| 0.02 pM Std | 0.02 pM Std | 0.02 pM Std | ||||
E | ALF_END_ |
PLT2_16S_3B | ALF_END_PLT4_ |
16S_ |
3B | ALF_END_ |
PLT6_16S_3B | ALF_END_PLT8_ |
16S_ |
3B |
| 0. |
2 pM Std | 0. |
2 pM Std | 0. |
2 pM Std |
F | ALF_END_ |
PLT2_16S |
_9H | ALF_END_ |
PLT4_16S |
_9H | ALF_END_ |
PLT6_16S |
_9H | ALF_END_ |
PLT8_16S |
_9H |
|
2 pM Std |
2 pM Std |
2 pM Std |
G | ALF_END_ |
ALF_END_PLT1_ITS
PLT2_ITS_3B | ALF_END_ |
PLT4_ITS |
_3B | ALF_END_ |
PLT6_ITS |
_3B | ALF_END_ |
PLT8_ITS_3B |
|
20 pM Std |
20 pM Std |
20 pM Std |
H | ALF_END_PLT2_ |
ITS_ |
9H | ALF_END_ |
PLT4_ITS_9H | ALF_END_ |
PLT6_ITS |
_9H | ALF_END_ |
PLT8_ITS |
ALF_END_PLT4_16S
_9H |
|
|
2 pM Std
2 pM Std
2 pM Std
|
|
Results:
Average results for the following plates:
ALF_END_PLT1_16S: 8.11 nanomoles
ALF_END_PLT1_ITS: 7.68 nanomoles
ALF_END_PLT2_16S: 7.4 nanomoles
ALF_END_PLT2_ITS: 9.16 nanomoles
ALF_END_PLT3_16S: 8.82 nanomoles
ALF_END_PLT3_ITS: 9.80 nanomoles
ALF_END_PLT4_16S
...
: 8.52 nanomoles
ALF_END_PLT4_ITS
...
...
20 pM Std
...
20 pM Std
...
20 pM Std
...
H
: 8.21 nanomoles
ALF_END_
...
PLT5_16S: 7.48 nanomoles
ALF_END_
...
PLT5_ITS: 7.32 nanomoles
ALF_END_
...
PLT6_16S: 6.89 nanomoles
ALF_END_
...
PLT6_ITS: 8.22 nanomoles
ALF_END_
...
PLT7_16S: 7.02 nanomoles
ALF_END_
...
PLT7_ITS: 8.03 nanomoles
ALF_END_
...
PLT8_16S: 7.21 nanomoles
ALF_END_
...
PLT8_ITS
...
...
...
...
Results:
Average results for the following plates (column 3):: 8.71 nanomoles
The full result report can be viewed below:
| View file | ||
|---|---|---|
|
...
|
...
|
...
ALF_END_PLT2_16S:
ALF_END_PLT2_ITS:
ALF_END_PLT3_16S:
ALF_END_PLT3_ITS:
ALF_END_PLT4_16S:
ALF_END_PLT4_ITS:
The full result report can be viewed below:
MasterMix (make for 16 plates in 50mL conical tube)
...
ul/rxn
...
Reagent
...
# of rxns
...
|
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 | Ultra Pure H2O | 1700 |
12,325 | ||
11 | Total Volume | 1700 |
18,700 |
Add 11 ul to each well of a hard shell, full skirt plate. Seal with bubble strips and store in refrigerator until needed label “ALF PCR”. (If doing manually, one needs 2 ul of template and 2 ul of the primers).
Plates | 16S MIDs | ITS MIDs |
|---|---|---|
ALF_ |
PLT1_2020_ |
EPI |
long16S0A3 |
longITS0A3 |
long16S0B3 |
longITS0B3 |
ALF_ |
PLT2_2020_ |
EPI |
long16S0C3 |
longITS0C3 |
long16S0D3 |
longITS0D3 |
ALF_ |
PLT3_2020_ |
EPI |
long16S0E3 |
longITS0E3 |
long16S0F3 |
longITS0F3 |
ALF_ |
PLT4_2020_ |
EPI |
long16S0G3 |
longITS0G3 |
long16S0H3 |
longITS0H3 |
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 ALF_END_RUN2
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
...
ALF_END_PLT5_16S
...
ALF_END_PLT5_ITS
...
ALF_END_PLT6_16S
...
ALF_END_PLT6_ITS
...
ALF_END_PLT7_16S
...
ALF_END_PLT7_ITS
...
ALF_END_PLT8_16S
...
ALF_END_PLT8_ITS
...
...
...
...
NTC
...
B
...
ALF_END_PLT5_16S
...
ALF_END_PLT5_ITS
...
ALF_END_PLT6_16S
...
ALF_END_PLT6_ITS
...
ALF_END_PLT7_16S
...
ALF_END_PLT7_ITS
...
ALF_END_PLT8_16S
...
ALF_END_PLT8_ITS
...
...
...
04.0002 pM Std
...
0.0002 pM Std
...
C
...
ALF_END_PLT5_16S
...
ALF_END_PLT5_ITS
...
ALF_END_PLT6_16S
...
ALF_END_PLT6_ITS
...
ALF_END_PLT7_16S
...
ALF_END_PLT7_ITS
...
ALF_END_PLT8_16S
...
ALF_END_PLT8_ITS
...
...
...
0.002 pM Std
...
0.002 pM Std
...
D
...
ALF_END_PLT5_16S
...
ALF_END_PLT5_ITS
...
ALF_END_PLT6_16S
...
ALF_END_PLT6_ITS
...
ALF_END_PLT7_16S
...
ALF_END_PLT7_ITS
...
ALF_END_PLT8_16S
...
ALF_END_PLT8_ITS
...
...
...
0.02 pM Std
...
0.02 pM Std
...
E
...
ALF_END_PLT5_16S
...
ALF_END_PLT5_ITS
...
ALF_END_PLT6_16S
...
ALF_END_PLT6_ITS
...
ALF_END_PLT7_16S
...
ALF_END_PLT7_ITS
...
ALF_END_PLT8_16S
...
ALF_END_PLT8_ITS
...
...
...
0.2 pM Std
...
0.2 pM Std
...
F
...
ALF_END_PLT5_16S
...
ALF_END_PLT5_ITS
...
ALF_END_PLT6_16S
...
ALF_END_PLT6_ITS
...
ALF_END_PLT7_16S
...
ALF_END_PLT7_ITS
...
ALF_END_PLT8_16S
...
ALF_END_PLT8_ITS
...
...
...
2 pM Std
...
2 pM Std
...
G
...
ALF_END_PLT5_16S
...
ALF_END_PLT5_ITS
...
ALF_END_PLT6_16S
...
ALF_END_PLT6_ITS
...
ALF_END_PLT7_16S
...
ALF_END_PLT7_ITS
...
ALF_END_PLT8_16S
...
ALF_END_PLT8_ITS
...
...
...
20 pM Std
...
20 pM Std
...
H
...
ALF_END_PLT5_16S
...
ALF_END_PLT5_ITS
...
ALF_END_PLT6_16S
...
ALF_END_PLT6_ITS
...
ALF_END_PLT7_16S
...
ALF_END_PLT7_ITS
...
ALF_END_PLT8_16S
...
ALF_END_PLT8_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
...
ALF_END_PLT5_16S
...
ALF_END_PLT5_ITS
...
ALF_END_PLT6_16S
...
ALF_END_PLT6_ITS
...
ALF_END_PLT7_16S
...
ALF_END_PLT7_ITS
...
ALF_END_PLT8_16S
...
ALF_END_PLT8_ITS
...
...
NTC
...
NTC
...
NTC
...
B
...
ALF_END_PLT5_16S
...
ALF_END_PLT5_ITS
...
ALF_END_PLT6_16S
...
ALF_END_PLT6_ITS
...
ALF_END_PLT7_16S
...
ALF_END_PLT7_ITS
...
ALF_END_PLT8_16S
...
ALF_END_PLT8_ITS
...
...
0.0002 pM Std
...
0.0002 pM Std
...
0.0002 pM Std
...
C
...
ALF_END_PLT5_16S
...
ALF_END_PLT5_ITS
...
ALF_END_PLT6_16S
...
ALF_END_PLT6_ITS
...
ALF_END_PLT7_16S
...
ALF_END_PLT7_ITS
...
ALF_END_PLT8_16S
...
ALF_END_PLT8_ITS
...
...
0.002 pM Std
...
0.002 pM Std
...
0.002 pM Std
...
D
...
ALF_END_PLT5_16S
...
ALF_END_PLT5_ITS
...
ALF_END_PLT6_16S
...
ALF_END_PLT6_ITS
...
ALF_END_PLT7_16S
...
ALF_END_PLT7_ITS
...
ALF_END_PLT8_16S
...
ALF_END_PLT8_ITS
...
...
0.02 pM Std
...
0.02 pM Std
...
0.02 pM Std
...
E
...
ALF_END_PLT5_16S
...
ALF_END_PLT5_ITS
...
ALF_END_PLT6_16S
...
ALF_END_PLT6_ITS
...
ALF_END_PLT7_16S
...
ALF_END_PLT7_ITS
...
ALF_END_PLT8_16S
...
ALF_END_PLT8_ITS
...
...
0.2 pM Std
...
0.2 pM Std
...
0.2 pM Std
...
F
...
ALF_END_PLT5_16S
...
ALF_END_PLT5_ITS
...
ALF_END_PLT6_16S
...
ALF_END_PLT6_ITS
...
ALF_END_PLT7_16S
...
ALF_END_PLT7_ITS
...
ALF_END_PLT8_16S
...
ALF_END_PLT8_ITS
...
...
2 pM Std
...
2 pM Std
...
2 pM Std
...
G
...
ALF_END_PLT5_16S
...
ALF_END_PLT5_ITS
...
ALF_END_PLT6_16S
...
ALF_END_PLT6_ITS
...
ALF_END_PLT7_16S
...
ALF_END_PLT7_ITS
...
ALF_END_PLT8_16S
...
ALF_END_PLT8_ITS
...
...
20 pM Std
...
20 pM Std
...
20 pM Std
...
H
...
ALF_END_PLT5_16S
...
ALF_END_PLT5_ITS
...
ALF_END_PLT6_16S
...
ALF_END_PLT6_ITS
...
ALF_END_PLT7_16S
...
ALF_END_PLT7_ITS
...
ALF_END_PLT8_16S
...
ALF_END_PLT8_ITS
...
...
...
...
Results:
Average results for the following plates (column 3):
ALF_END_PLT5_16S:
ALF_END_PLT5_ITS:
ALF_END_PLT6_16S:
ALF_END_PLT6_ITS:
ALF_END_PLT7_16S:
ALF_END_PLT7_ITS:
ALF_END_PLT8_16S:
ALF_END_PLT8_ITS:
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
...
Ultra Pure 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 “ALF PCR”. (If doing manually, one needs 2 ul of template and 2 ul of the primers).
...
Plates
...
16S MIDs
...
ITS MIDs
...
ALF_PLT1_2020_EPI
...
long16S0A3
...
longITS0A3
...
long16S0B3
...
longITS0B3
...
ALF_PLT2_2020_EPI
...
long16S0C3
...
longITS0C3
...
long16S0D3
...
longITS0D3
...
ALF_PLT3_2020_EPI
...
long16S0E3
...
longITS0E3
...
long16S0F3
...
longITS0F3
...
ALF_PLT4_2020_EPI
...
long16S0G3
...
longITS0G3
...
long16S0H3
...
longITS0H3
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 ALF_EPI_RUN1
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
...
ALF_EPI_PLT1_16S
...
ALF_EPI_PLT1_ITS
...
ALF_EPI_PLT2_16S
...
ALF_EPI_PLT2_ITS
...
ALF_EPI_PLT3_16S
...
ALF_EPI_PLT3_ITS
...
ALF_EPI_PLT4_16S
...
ALF_EPI_PLT4_ITS
...
...
...
...
NTC
...
B
...
ALF_EPI_PLT1_16S
...
ALF_EPI_PLT1_ITS
...
ALF_EPI_PLT2_16S
...
ALF_EPI_PLT2_ITS
...
ALF_EPI_PLT3_16S
...
ALF_EPI_PLT3_ITS
...
ALF_EPI_PLT4_16S
...
ALF_EPI_PLT4_ITS
...
...
...
04.0002 pM Std
...
0.0002 pM Std
...
C
...
ALF_EPI_PLT1_16S
...
ALF_EPI_PLT1_ITS
...
ALF_EPI_PLT2_16S
...
ALF_EPI_PLT2_ITS
...
ALF_EPI_PLT3_16S
...
ALF_EPI_PLT3_ITS
...
ALF_EPI_PLT4_16S
...
ALF_EPI_PLT4_ITS
...
...
...
0.002 pM Std
...
0.002 pM Std
...
D
...
ALF_EPI_PLT1_16S
...
ALF_EPI_PLT1_ITS
...
ALF_EPI_PLT2_16S
...
ALF_EPI_PLT2_ITS
...
ALF_EPI_PLT3_16S
...
ALF_EPI_PLT3_ITS
...
ALF_EPI_PLT4_16S
...
ALF_EPI_PLT4_ITS
...
...
...
0.02 pM Std
...
0.02 pM Std
...
E
...
ALF_EPI_PLT1_16S
...
ALF_EPI_PLT1_ITS
...
ALF_EPI_PLT2_16S
...
ALF_EPI_PLT2_ITS
...
ALF_EPI_PLT3_16S
...
ALF_EPI_PLT3_ITS
...
ALF_EPI_PLT4_16S
...
ALF_EPI_PLT4_ITS
...
...
...
0.2 pM Std
...
0.2 pM Std
...
F
...
ALF_EPI_PLT1_16S
...
ALF_EPI_PLT1_ITS
...
ALF_EPI_PLT2_16S
...
ALF_EPI_PLT2_ITS
...
ALF_EPI_PLT3_16S
...
ALF_EPI_PLT3_ITS
...
ALF_EPI_PLT4_16S
...
ALF_EPI_PLT4_ITS
...
...
...
2 pM Std
...
2 pM Std
...
G
...
ALF_EPI_PLT1_16S
...
ALF_EPI_PLT1_ITS
...
ALF_EPI_PLT2_16S
...
ALF_EPI_PLT2_ITS
...
ALF_EPI_PLT3_16S
...
ALF_EPI_PLT3_ITS
...
ALF_EPI_PLT4_16S
...
ALF_EPI_PLT4_ITS
...
...
...
20 pM Std
...
20 pM Std
...
H
...
ALF_EPI_PLT1_16S
...
ALF_EPI_PLT1_ITS
...
ALF_EPI_PLT2_16S
...
ALF_EPI_PLT2_ITS
...
ALF_EPI_PLT3_16S
...
ALF_EPI_PLT3_ITS
...
ALF_EPI_PLT4_16S
...
ALF_EPI_PLT4_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
...
ALF_EPI_PLT1_16S
...
ALF_EPI_PLT1_ITS
...
ALF_EPI_PLT2_16S
...
ALF_EPI_PLT2_ITS
...
ALF_EPI_PLT3_16S
...
ALF_EPI_PLT3_ITS
...
ALF_EPI_PLT4_16S
...
ALF_EPI_PLT4_ITS
...
...
NTC
...
NTC
...
NTC
...
B
...
ALF_EPI_PLT1_16S
...
ALF_EPI_PLT1_ITS
...
ALF_EPI_PLT2_16S
...
ALF_EPI_PLT2_ITS
...
ALF_EPI_PLT3_16S
...
ALF_EPI_PLT3_ITS
...
ALF_EPI_PLT4_16S
...
ALF_EPI_PLT4_ITS
...
...
0.0002 pM Std
...
0.0002 pM Std
...
0.0002 pM Std
...
C
...
ALF_EPI_PLT1_16S
...
ALF_EPI_PLT1_ITS
...
ALF_EPI_PLT2_16S
...
ALF_EPI_PLT2_ITS
...
ALF_EPI_PLT3_16S
...
ALF_EPI_PLT3_ITS
...
ALF_EPI_PLT4_16S
...
ALF_EPI_PLT4_ITS
...
...
0.002 pM Std
...
0.002 pM Std
...
0.002 pM Std
...
D
...
ALF_EPI_PLT1_16S
...
ALF_EPI_PLT1_ITS
...
ALF_EPI_PLT2_16S
...
ALF_EPI_PLT2_ITS
...
ALF_EPI_PLT3_16S
...
ALF_EPI_PLT3_ITS
...
ALF_EPI_PLT4_16S
...
ALF_EPI_PLT4_ITS
...
...
0.02 pM Std
...
0.02 pM Std
...
0.02 pM Std
...
E
...
ALF_EPI_PLT1_16S
...
ALF_EPI_PLT1_ITS
...
ALF_EPI_PLT2_16S
...
ALF_EPI_PLT2_ITS
...
ALF_EPI_PLT3_16S
...
ALF_EPI_PLT3_ITS
...
ALF_EPI_PLT4_16S
...
ALF_EPI_PLT4_ITS
...
...
0.2 pM Std
...
0.2 pM Std
...
0.2 pM Std
...
F
...
ALF_EPI_PLT1_16S
...
ALF_EPI_PLT1_ITS
...
ALF_EPI_PLT2_16S
...
ALF_EPI_PLT2_ITS
...
ALF_EPI_PLT3_16S
...
ALF_EPI_PLT3_ITS
...
ALF_EPI_PLT4_16S
...
ALF_EPI_PLT4_ITS
...
...
2 pM Std
...
2 pM Std
...
2 pM Std
...
G
...
ALF_EPI_PLT1_16S
...
ALF_EPI_PLT1_ITS
...
ALF_EPI_PLT2_16S
...
ALF_EPI_PLT2_ITS
...
ALF_EPI_PLT3_16S
...
ALF_EPI_PLT3_ITS
...
ALF_EPI_PLT4_16S
...
ALF_EPI_PLT4_ITS
...
...
20 pM Std
...
20 pM Std
...
20 pM Std
...
H
...
ALF_EPI_PLT1_16S
...
ALF_EPI_PLT1_ITS
...
ALF_EPI_PLT2_16S
...
ALF_EPI_PLT2_ITS
...
ALF_EPI_PLT3_16S
...
ALF_EPI_PLT3_ITS
...
ALF_EPI_PLT4_16S
...
ALF_EPI_PLT4_ITS
...
...
...
...
Results:
Average results for the following plates (column 3):
ALF_EPI_PLT1_16S:
ALF_EPI_PLT1_ITS:
ALF_EPI_PLT2_16S:
ALF_EPI_PLT2_ITS:
ALF_EPI_PLT3_16S:
ALF_EPI_PLT3_ITS:
ALF_EPI_PLT4_16S:
ALF_EPI_PLT4_ITS:
The full result report can be viewed below:
MasterMix (make for 16 plates 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 | Ultra Pure H2O | 1700 | 12,325 |
11 | Total Volume | 1700 | 18,700 |
Add 11 ul to each well of a hard shell, full skirt plate. Seal with bubble strips and store in refrigerator until needed label “ALF PCR”. (If doing manually, one needs 2 ul of template and 2 ul of the primers).
Plates | 16S MIDs | ITS MIDs |
|---|---|---|
ALF_PLT5_2020_EPI | long16S0A4 | longITS0A4 |
long16S0B4 | longITS0B4 | |
ALF_PLT6_2020_EPI | long16S0C4 | longITS0C4 |
long16S0D4 | longITS0D4 | |
ALF_PLT7_2020_EPI | long16S0E4 | longITS0E4 |
long16S0F4 | longITS0F4 | |
ALF_PLT8_2020_EPI | long16S0G4 | longITS0G4 |
long16S0H4 | longITS0H4 |
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 ALF_EPI
Make 1:1000 dilutions 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 | ALF_EPI_PLT1_16S_3B | ALF_EPI_PLT3_16S_3B | ALF_EPI_PLT5_16S_3B | ALF_EPI_PLT7_16S_3B |
| NTC | NTC | NTC | ||||
B | ALF_EPI_PLT1_16S_9H | ALF_EPI_PLT3_16S_9H | ALF_EPI_PLT5_16S_9H | ALF_EPI_PLT7_16S_9H |
| 0.0002 pM Std | 0.0002 pM Std | 0.0002 pM Std | ||||
C | ALF_EPI_PLT1_ITS_3B | ALF_EPI_PLT3_ITS_3B | ALF_EPI_PLT5_ITS_3B | ALF_EPI_PLT7_ITS_3B |
| 0.002 pM Std | 0.002 pM Std | 0.002 pM Std | ||||
D | ALF_EPI_PLT1_ITS_9H | ALF_EPI_PLT3_ITS_9H | ALF_EPI_PLT5_ITS_9H | ALF_EPI_PLT7_ITS_9H |
| 0.02 pM Std | 0.02 pM Std | 0.02 pM Std | ||||
E | ALF_EPI_PLT2_16S_3B | ALF_EPI_PLT4_16S_3B | ALF_EPI_PLT6_16S_3B | ALF_EPI_PLT8_16S_3B |
| 0.2 pM Std | 0.2 pM Std | 0.2 pM Std | ||||
F | ALF_EPI_PLT2_16S_9H | ALF_EPI_PLT4_16S_9H | ALF_EPI_PLT6_16S_9H | ALF_EPI_PLT8_16S_9H |
| 2 pM Std | 2 pM Std | 2 pM Std | ||||
G | ALF_EPI_PLT2_ITS_3B | ALF_EPI_PLT4_ITS_3B | ALF_EPI_PLT6_ITS_3B | ALF_EPI_PLT8_ITS_3B |
| 20 pM Std | 20 pM Std | 20 pM Std | ||||
H | ALF_EPI_PLT2_ITS_9H | ALF_EPI_PLT4_ITS_9H | ALF_EPI_PLT6_ITS_9H | ALF_EPI_PLT8_ITS_9H |
|
|
|
|
Add 16 ul of Illumina Library Quantification MasterMix to each well:
ul/rxn | Reagent | # of rxns | ul needed |
|---|---|---|---|
10 ul | KAPA SYBR FAST qPCR MM (2X) | 60 | 600 |
2 ul | Primer Premix (10X) | 60 | 120 |
4 ul | Ultra Pure Water | 60 | 240 |
16 ul | Total Volume | 60 | 960 |
Add 4 ul of template, pool, or standards to each well:
Results:
Average results for the following plates:
ALF_EPI_PLT1_16S: 12.31 nanomoles
ALF_EPI_PLT1_ITS: 12.34 nanomoles
ALF_EPI_PLT2_16S: 2.79 nanomoles
ALF_EPI_PLT2_ITS: 11.35 nanomoles
ALF_EPI_PLT3_16S: 33.59 nanomoles
ALF_EPI_PLT3_ITS: 31.96 nanomoles
ALF_EPI_PLT4_16S: 4.40 nanomoles
ALF_EPI_PLT4_ITS: 10.99 nanomoles
ALF_EPI_PLT5_16S: 0.47 nanomoles
ALF_EPI_PLT5_ITS: 0.89 nanomoles
ALF_EPI_PLT6_16S: 2.23 nanomoles
ALF_EPI_PLT6_ITS: 4.86 nanomoles
ALF_EPI_PLT7_16S: 3.98 nanomoles
ALF_EPI_PLT7_ITS: 26.33 nanomoles
ALF_EPI_PLT8_16S: 28.78 nanomoles
ALF_EPI_PLT8_ITS: 35.00 nanomoles
The full result report can be viewed 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 | Kapa HiFi HotStart DNA Pol | 1700 | 510 |
7.25 | Ultra Pure H2O | 1700 | 12,325 |
11 | Total Volume | 1700 | 18,700 |
Add 11 ul to each well of a hard shell, full skirt plate. Seal with bubble strips and store in refrigerator until needed label “ALF PCR”. (If doing manually, one needs 2 ul of template and 2 ul of the primers).
Plates | 16S MIDs | ITS MIDs |
|---|---|---|
ALF_PLT1_2017_GBS | long16S0A5 | longITS0A5 |
long16S0B5 | longITS0B5 | |
ALF_PLT2_2017_GBS | long16S0C5 | longITS0C5 |
long16S0D5 | longITS0D5 | |
ALF_PLT3_2017_GBS | long16S0E5 | longITS0E5 |
long16S0F5 | longITS0F5 | |
ALF_PLT4_2017_GBS | long16S0G5 | longITS0G5 |
long16S0H5 | longITS0H5 |
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)
MasterMix (make for 12 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 | Ultra Pure H2O | 1700 | 12,325 |
11 | Total Volume | 1700 | 18,700 |
Add 11 ul to each well of a hard shell, full skirt plate. Seal with bubble strips and store in refrigerator until needed label “ALF PCR”. (If doing manually, one needs 2 ul of template and 2 ul of the primers).
Plates | 16S MIDs | ITS MIDs |
|---|---|---|
ALF_PLT5_2017_GBS | long16S0A6 | longITS0A6 |
long16S0B6 | longITS0B6 | |
ALF_PLT6_2017_GBS | long16S0C6 | longITS0C6 |
long16S0D6 | longITS0D6 | |
ALF_PLT7_2017_GBS | long16S0E6 | longITS0E6 |
long16S0F6 | longITS0F6 | |
ALF_PLT8_2017_GBS | long16S0G6 | longITS0G6 |
long16S0H6 | longITS0H6 |
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)
MasterMix (in 50mL conical tube)
ul/rxn | Reagent | # of rxns | ul needed |
|---|---|---|---|
3 | 5X Kapa HiFi Buffer |
1550 |
4650 | |
0.45 | 10M dNTPs |
1550 |
697.5 | |
0.3 | Kapa HiFi HotStart DNA Pol |
1550 |
465 | |
7.25 | Ultra Pure H2O |
1700
1550 | 11,237.5 |
11 | Total Volume |
1550 |
17,050 |
Add 11 ul to each well of a hard shell, full skirt plate. Seal with bubble strips and store in refrigerator until needed label “ALF PCR”. (If doing manually, one needs 2 ul of template and 2 ul of the primers).
Plates | 16S MIDs | ITS MIDs |
|---|---|---|
ALF |
long16S0A4
longITS0A4
long16S0B4
longITS0B4
ALF_PLT6_2020_EPI
long16S0C4
longITS0C4
long16S0D4
longITS0D4
_PLT9_2017_GBS | long16S0A7 | longITS0A7 |
long16S0B7 | longITS0B7 | |
ALF_PLT10_2017_GBS | long16S0C7 | longITS0C7 |
long16S0D7 | longITS0D7 | |
ALF_PLT11_2017_GBS | long16S0E7 | longITS0E7 |
long16S0F7 | longITS0F7 | |
ALF_PLT9_2020_EPI |
long16S0G7 |
longITS0G7 |
long16S0H7 |
longITS0H7 |
ALF_ |
PLT1_2020_ |
SOIL |
long16S0A8 |
longITS0A8 |
long16S0B8 |
longITS0B8 |
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 ALF_
...
GBS
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 | ALF_ |
GBS_ |
PLT1_16S_3B | ALF_ |
GBS_PLT3_ |
16S_ |
3B | ALF_ |
GBS_ |
PLT5_16S |
_3B | ALF_ |
GBS_PLT7_16S_3B | ALF_ |
GBS_PLT9_ |
16S_ |
3B | ALF_ |
GBS_ |
PLT11_16S_3B | ALF_PLT1_2020_ |
SOIL_ |
16S_ |
3B |
| NTC | NTC | NTC | |
B | ALF_ |
GBS_ |
PLT1_16S_9H | ALF_GBS_ |
PLT3_ |
16S_ |
9H | ALF_ |
GBS_ |
PLT5_16S |
_9H | ALF_ |
GBS_PLT7_16S_9H | ALF_ |
GBS_PLT9_ |
16S_ |
9H | ALF_ |
GBS_ |
PLT11_16S_9A | ALF_PLT1_2020_ |
SOIL_ |
16S_ |
9H |
| 0.0002 pM Std | 0.0002 pM Std | 0.0002 pM Std | |
C | ALF_GBS_ |
PLT1_ |
ITS_ |
3B | ALF_ |
GBS_ |
PLT3_ITS |
_3B | ALF_ |
GBS_ |
PLT5_ITS_3B | ALF_ |
GBS_PLT7_ITS_ |
3B | ALF_ |
GBS_ |
PLT9_ITS_3B | ALF_GBS_ |
PLT11_ |
ITS_ |
3B | ALF_ |
PLT1_2020_ |
SOIL_ITS_3B |
| 0.002 pM Std | 0.002 pM Std | 0.002 pM Std | |
D | ALF_ |
GBS_PLT1_ |
ITS_ |
9H | ALF_ |
GBS_ |
PLT3_ITS |
_9H | ALF_ |
GBS_ |
PLT5_ITS_9H | ALF_ |
GBS_PLT7_ITS_ |
9H | ALF_ |
GBS_ |
PLT9_ITS_9H | ALF_ |
GBS_PLT11_ |
ITS_ |
9A | ALF_ |
PLT1_2020_ |
SOIL_ITS_9H |
| 0.02 pM Std | 0.02 pM Std | 0.02 pM Std | |
E | ALF_ |
GBS_ |
PLT2_16S |
_3B | ALF_ |
GBS_ |
PLT4_16S_3B | ALF_ |
GBS_PLT6_16S_ |
3B | ALF_ |
GBS_ |
PLT8_16S_3B | ALF_ |
GBS_PLT10_ |
16S_ |
3B | ALF_ |
PLT9_2020_EPI_ |
16S_ |
1B |
| 0.2 pM Std | 0.2 pM Std | 0.2 pM Std | ||
F | ALF_ |
GBS_ |
PLT2_16S_9H | ALF_ |
GBS_PLT4_ |
16S_ |
9H | ALF_ |
GBS_PLT6_16S_9H | ALF_GBS_ |
PLT8_ |
16S_ |
9H | ALF_ |
GBS_ |
PLT10_16S_9H | ALF_ |
PLT9_ |
2020_ |
EPI_ |
16S |
_3H |
| 2 pM Std | 2 pM Std | 2 pM Std | ||
G |
ALF_ |
GBS_ |
PLT2_ITS_3B | ALF_GBS_ |
PLT4_ |
ITS_ |
3B | ALF_ |
GBS_PLT6_ITS_3B | ALF_ |
GBS_PLT8_ |
ITS_ |
3B | ALF_ |
GBS_ |
PLT10_ITS_3B | ALF_ |
PLT9_2020_EPI_ |
ITS_ |
1B |
| 20 pM Std | 20 pM Std | 20 pM Std | ||
H | ALF_GBS_ |
PLT2_ |
ITS_ |
9H | ALF_ |
GBS_ |
PLT4_ITS_9H | ALF_ |
GBS_PLT6_ITS_ |
9H | ALF_ |
GBS_ |
PLT8_ITS |
_9H | ALF_ |
GBS_ |
PLT10_ITS_9H | ALF_ |
PLT9_2020_EPI_ |
ITS_ |
3H |
|
|
|
|
Add 16 ul of Illumina Library Quantification MasterMix to each well:
Results:
Average results for the following plates (column 3):
ALF_EPI_PLT5_16S:
ALF_EPI_PLT5_ITS:
ALF_EPI_PLT6_16S:
ALF_EPI_PLT6_ITS:
ALF_EPI_PLT7_16S:
ALF_EPI_PLT7_ITS:
ALF_EPI_PLT8_16S:
...
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
...
ALF_EPI_PLT5_16S
...
ALF_EPI_PLT5_ITS
...
ALF_EPI_PLT6_16S
...
ALF_EPI_PLT6_ITS
...
ALF_EPI_PLT7_16S
...
ALF_EPI_PLT7_ITS
...
ALF_EPI_PLT8_16S
...
ALF_EPI_PLT8_ITS
...
...
NTC
...
NTC
...
NTC
...
B
...
ALF_EPI_PLT5_16S
...
ALF_EPI_PLT5_ITS
...
ALF_EPI_PLT6_16S
...
ALF_EPI_PLT6_ITS
...
ALF_EPI_PLT7_16S
...
ALF_EPI_PLT7_ITS
...
ALF_EPI_PLT8_16S
...
ALF_EPI_PLT8_ITS
...
...
0.0002 pM Std
...
0.0002 pM Std
...
0.0002 pM Std
...
C
...
ALF_EPI_PLT5_16S
...
ALF_EPI_PLT5_ITS
...
ALF_EPI_PLT6_16S
...
ALF_EPI_PLT6_ITS
...
ALF_EPI_PLT7_16S
...
ALF_EPI_PLT7_ITS
...
ALF_EPI_PLT8_16S
...
ALF_EPI_PLT8_ITS
...
...
0.002 pM Std
...
0.002 pM Std
...
0.002 pM Std
...
D
...
ALF_EPI_PLT5_16S
...
ALF_EPI_PLT5_ITS
...
ALF_EPI_PLT6_16S
...
ALF_EPI_PLT6_ITS
...
ALF_EPI_PLT7_16S
...
ALF_EPI_PLT7_ITS
...
ALF_EPI_PLT8_16S
...
ALF_EPI_PLT8_ITS
...
...
0.02 pM Std
...
0.02 pM Std
...
0.02 pM Std
...
E
...
ALF_EPI_PLT5_16S
...
ALF_EPI_PLT5_ITS
...
ALF_EPI_PLT6_16S
...
ALF_EPI_PLT6_ITS
...
ALF_EPI_PLT7_16S
...
ALF_EPI_PLT7_ITS
...
ALF_EPI_PLT8_16S
...
ALF_EPI_PLT8_ITS
...
...
0.2 pM Std
...
0.2 pM Std
...
0.2 pM Std
...
F
...
ALF_EPI_PLT5_16S
...
ALF_EPI_PLT5_ITS
...
ALF_EPI_PLT6_16S
...
ALF_EPI_PLT6_ITS
...
ALF_EPI_PLT7_16S
...
ALF_EPI_PLT7_ITS
...
ALF_EPI_PLT8_16S
...
ALF_EPI_PLT8_ITS
...
...
2 pM Std
...
2 pM Std
...
2 pM Std
...
G
...
ALF_EPI_PLT5_16S
...
ALF_EPI_PLT5_ITS
...
ALF_EPI_PLT6_16S
...
ALF_EPI_PLT6_ITS
...
ALF_EPI_PLT7_16S
...
ALF_EPI_PLT7_ITS
...
ALF_EPI_PLT8_16S
...
ALF_EPI_PLT8_ITS
...
...
20 pM Std
...
20 pM Std
...
20 pM Std
...
H
...
ALF_EPI_PLT5_16S
...
ALF_EPI_PLT5_ITS
...
ALF_EPI_PLT6_16S
...
ALF_EPI_PLT6_ITS
...
ALF_EPI_PLT7_16S
...
ALF_EPI_PLT7_ITS
...
ALF_EPI_PLT8_16S
...
ALF_EPI_PLT8_ITS
...
...
...
...
90 | 600 | ||
2 ul | Primer Premix (10X) | 90 | 120 |
4 ul | Ultra Pure Water | 90 | 240 |
16 ul | Total Volume | 90 | 960 |
Add 4 ul of template, pool, or standards to each well:
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
A | ALF_GBS_PLT1_16S_3B | ALF_GBS_PLT3_16S_3B | ALF_GBS_PLT5_16S_3B | ALF_GBS_PLT7_16S_3B | ALF_GBS_PLT9_16S_3B | ALF_GBS_PLT11_16S_3B | ALF_PLT1_2020_SOIL_16S_3B | TRNL_16S_Pool |
| NTC | NTC | NTC |
B | ALF_GBS_PLT1_16S_9H | ALF_GBS_PLT3_16S_9H | ALF_GBS_PLT5_16S_9H | ALF_GBS_PLT7_16S_9H | ALF_GBS_PLT9_16S_9H | ALF_GBS_PLT11_16S_9A | ALF_PLT1_2020_SOIL_16S_9H | TRNL_16S_Pool |
| 0.0002 pM Std | 0.0002 pM Std | 0.0002 pM Std |
C | ALF_GBS_PLT1_ITS_3B | ALF_GBS_PLT3_ITS_3B | ALF_GBS_PLT5_ITS_3B | ALF_GBS_PLT7_ITS_3B | ALF_GBS_PLT9_ITS_3B | ALF_GBS_PLT11_ITS_3B | ALF_PLT1_2020_SOIL_ITS_3B |
| 0.002 pM Std | 0.002 pM Std | 0.002 pM Std | |
D | ALF_GBS_PLT1_ITS_9H | ALF_GBS_PLT3_ITS_9H | ALF_GBS_PLT5_ITS_9H | ALF_GBS_PLT7_ITS_9H | ALF_GBS_PLT9_ITS_9H | ALF_GBS_PLT11_ITS_9A | ALF_PLT1_2020_SOIL_ITS_9H |
| 0.02 pM Std | 0.02 pM Std | 0.02 pM Std | |
E | ALF_GBS_PLT2_16S_3B | ALF_GBS_PLT4_16S_3B | ALF_GBS_PLT6_16S_3B | ALF_GBS_PLT8_16S_3B | ALF_GBS_PLT10_16S_3B | ALF_PLT9_2020_EPI_16S_1B | TRNL_T_Pool |
| 0.2 pM Std | 0.2 pM Std | 0.2 pM Std | |
F | ALF_GBS_PLT2_16S_9H | ALF_GBS_PLT4_16S_9H | ALF_GBS_PLT6_16S_9H | ALF_GBS_PLT8_16S_9H | ALF_GBS_PLT10_16S_9H | ALF_PLT9_2020_EPI_16S_3H | TRNL_T_Pool |
| 2 pM Std | 2 pM Std | 2 pM Std | |
G | ALF_GBS_PLT2_ITS_3B | ALF_GBS_PLT4_ITS_3B | ALF_GBS_PLT6_ITS_3B | ALF_GBS_PLT8_ITS_3B | ALF_GBS_PLT10_ITS_3B | ALF_PLT9_2020_EPI_ITS_1B | TRNL_T_Pool |
| 20 pM Std | 20 pM Std | 20 pM Std | |
H | ALF_GBS_PLT2_ITS_9H | ALF_GBS_PLT4_ITS_9H | ALF_GBS_PLT6_ITS_9H | ALF_GBS_PLT8_ITS_9H | ALF_GBS_PLT10_ITS_9H | ALF_PLT9_2020_EPI_ITS_3H | TRNL_16S_Pool |
Results:
Average results for the following plates:
ALF_GBS_PLT1_16S: 11.05 nanomoles
ALF_GBS_PLT1_ITS: 30.57 nanomoles
ALF_GBS_PLT2_16S: 14.58 nanomoles
ALF_GBS_PLT2_ITS: 6.37 nanomoles
ALF_GBS_PLT3_16S: 18.19 nanomoles
ALF_GBS_PLT3_ITS: 6.43 nanomoles
ALF_GBS_PLT4_16S: 6.71 nanomoles
ALF_GBS_PLT4_ITS: 5.98 nanomoles
ALF_GBS_PLT5_16S: 7.01 nanomoles
ALF_GBS_PLT5 ITS: 5.84 nanomoles
ALF_GBS_PLT6_16S: : 6.29 nanomoles
ALF_GBS_PLT6_ITS: 1.22 nanomoles
ALF_GBS_PLT7_16S: 1.96 nanomoles
ALF_GBS_PLT7_ITS: 2.77 nanomoles
ALF_GBS_PLT8_16S: 9.72 nanomoles
ALF_GBS_PLT8_ITS: 6.96 nanomoles
ALF_GBS_PLT9_16S: 13.38 nanomoles
ALF_GBS_PLT9_ITS: 15.33 nanomoles
ALF_GBS_PLT10_16S: 8.09 nanomoles
ALF_GBS_PLT10_ITS: 8.1 nanomoles
ALF_GBS_PLT11_16S: 6.30 nanomoles
ALF_GBS_PLT11_ITS: 7.88 nanomoles
ALF_PLT9_2020_EPI_16S: 1.69 nanomoles
ALF_PLT9_2020_EPI_ITS: 0.677 nanomoles
ALF_PLT1_2020_SOIL_16S: 3.29 nanomoles
ALF_PLT1_2020_SOIL_ITS: 12.42 nanomoles
The full result report can be viewed below:
| View file | ||
|---|---|---|
|