*Make Protocol Below for 2017 GBS Plates….
Prepare two ITS and two 16S PCR plates for the following template plates:
...
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 | 170012325 | 12,325 |
11 | Total Volume | 170018700 | 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).
...
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
...
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_9E | ALF_END_PLT1PLT3_16S_ITS9E | ALF_END_PLT2PLT5_16SALF_END_PLT2_ITS_9E | ALF_END_PLT3PLT7_16S | ALF_END_PLT3_ITS | ALF_END_PLT4_16S | ALF_END_PLT4_ITS |
| 2 pM Std | 2 pM Std | 2 pM Std | G_9E |
|
| 04.0002 pM Std | 0.0002 pM Std | ||||
C | ALF_END_PLT1_ITS_16S3B | ALF_END_PLT1PLT3_ITS_3B | ALF_END_PLT5_PLT2ITS_16S3B | ALF_END_PLT2PLT7_ITS_3B |
|
| 0.002 pM Std | 0.002 pM Std | ||||||||||||
D | ALF_END_PLT1_PLT3ITS_16S9E | ALF_END_PLT3_ITS_9E | ALF_END_PLT5_PLT4ITS_16S9E | ALF_END_PLT4PLT7_ITS_9E |
| 20 pM Std | 20 | 0.02 pM Std | 20 0.02 pM Std | |||||||||||
HE | ALF_END_PLT1PLT2_16S_3B | ALF_END_PLT1PLT4_16S_ITS3B | ALF_END_PLT2PLT6_16S_3B | ALF_END_PLT2_ITSPLT8_16S_3B |
|
| 0.2 pM Std | 0.2 pM Std | ||||||||||||
F | ALF_END_PLT3PLT2_16S_9E | ALF_END_PLT3PLT4_16S_ITS9E | ALF_END_PLT4PLT6_16S_9E | ALF_END_PLT4PLT8_16S_ITS9E |
|
|
|
|
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 | 2002 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_9E | ALF_END_PLT4_ITS_9E | ALF_END_PLT6_ITS_9E | ALF_END_PLT8_ITS_9E |
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 | 10060 | 400240 |
16 ul | Total Volume | 10060 | 1600960 |
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 |
_9E | ALF_END_ |
PLT3_16S |
_9E | ALF_END_ |
PLT5_16S |
_9E | ALF_END_ |
PLT7_16S |
_9E |
| 0.0002 pM Std | 0.0002 pM Std | 0.0002 pM Std | ||||
C | ALF_END_PLT1_ |
ITS_3B | ALF_END_ |
PLT3_ITS_3B | ALF_END_ |
PLT5_ITS_ |
3B | ALF_END_ |
PLT7_ITS |
ALF_END_PLT3_16S
ALF_END_PLT3_ITS
ALF_END_PLT4_16S
ALF_END_PLT4_ITS
_3B |
| 0.002 pM Std | 0.002 pM Std | 0.002 pM Std | ||||
D |
ALF_END_PLT1_ITS |
_9E | ALF_END_ |
PLT3_ITS |
_9E | ALF_END_ |
PLT5_ITS |
_9E | ALF_END_ |
PLT7_ITS_9E |
| 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_ |
ALF_END_PLT3_16S
ALF_END_PLT3_ITS
ALF_END_PLT4_16S
ALF_END_PLT4_ITS
16S_3B |
| 0.2 pM Std | 0.2 pM Std | 0.2 pM Std | ||||
F | ALF_END_ |
PLT2_16S |
_9E | ALF_END_ |
PLT4_16S |
_9E | ALF_END_ |
PLT6_16S |
_9E | ALF_END_ |
PLT8_16S |
_9E |
| 2 pM Std | 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 |
ALF_END_PLT3_16S
ALF_END_PLT3_ITS
ALF_END_PLT4_16S
ALF_END_PLT4_ITS
_3B |
| 20 pM Std | 20 pM Std | 20 pM Std | ||||
H | ALF_END_ |
ALF_END_PLT1_ITS
PLT2_ITS_9E | ALF_END_ |
PLT4_ITS |
_9E | ALF_END_ |
PLT6_ITS |
_9E | ALF_END_ |
PLT8_ITS_9E |
|
|
|
|
Results:
Average results for the following plates (column 3):
ALF_END_PLT1_16S:
ALF_END_PLT1_ITS:
...
ALF_END_PLT4_16S:
ALF_END_PLT4_ITS:
The full result report can 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 | 170012325 | 12,325 |
11 | Total Volume | 170018700 | 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_PLT5PLT1_2020_ENDEPI | long16S0A2 long16S0A3 | longITS0A2 longITS0A3 |
long16S0B2 long16S0B3 | longITS0B2 longITS0B3 | |
ALF_PLT6PLT2_2020_ENDEPI | long16S0C2 long16S0C3 | longITS0C2 longITS0C3 |
long16S0D2 long16S0D3 | longITS0D2 longITS0D3 | |
ALF_PLT7PLT3_2020_ENDEPI | long16S0E2 long16S0E3 | longITS0E2 longITS0E3 |
long16S0F2 long16S0F3 | longITS0F2 longITS0F3 | |
ALF_PLT8PLT4_2020_ENDEPI | long16S0G2 long16S0G3 | longITS0G2 longITS0G3 |
long16S0H2 long16S0H3 | longITS0H2 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
...
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_9E | ALF_EPI_PLT3_16S_9E | ALF_EPI_PLT5_16S_9E | ALF_EPI_PLT7_16S_9E |
| 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_9E | ALF_EPI_PLT3_ITS_9E | ALF_EPI_PLT5_ITS_9E | ALF_EPI_PLT7_ITS_9E |
| 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_9E | ALF_EPI_PLT4_16S_9E | ALF_EPI_PLT6_16S_9E | ALF_EPI_PLT8_16S_9E |
| 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_9E | ALF_EPI_PLT4_ITS_9E | ALF_EPI_PLT6_ITS_9E | ALF_EPI_PLT8_ITS_9E |
|
|
|
|
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_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_9E | ALF_EPI_PLT3_16S_9E | ALF_EPI_PLT5_16S_9E | ALF_EPI_PLT7_16S_9E |
| 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_9E | ALF_EPI_PLT3_ITS_9E | ALF_EPI_PLT5_ITS_9E | ALF_EPI_PLT7_ITS_9E |
| 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_9E | ALF_EPI_PLT4_16S_9E | ALF_EPI_PLT6_16S_9E | ALF_EPI_PLT8_16S_9E |
| 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_ |
9E | ALF_ |
EPI_ |
PLT4_ITS_9E | ALF_ |
EPI_PLT6_ITS_ |
9E | ALF_ |
EPI_ |
PLT8_ITS_9E |
Results:
Average results for the following plates:
ALF_
...
EPI_
...
PLT1_16S:
ALF_
...
EPI_
...
PLT1_ITS:
ALF_
...
EPI_
...
PLT2_16S:
ALF_
...
EPI_
...
PLT2_ITS
...
...
...
...
Results:
Average results for the following plates (column 3):
ALF_ENDEPI_PLT5PLT3_16S:
ALF_ENDEPI_PLT5PLT3_ITS:
ALF_ENDEPI_PLT6PLT4_16S:
ALF_ENDEPI_PLT6PLT4_ITS:
ALF_ENDEPI_PLT7PLT5_16S:
ALF_ENDEPI_PLT7PLT5_ITS:
ALF_ENDEPI_PLT8PLT6_16S:
ALF_ENDEPI_PLT8PLT6_ITS:
The full result ALF_EPI_PLT7_16S:
ALF_EPI_PLT7_ITS:
ALF_EPI_PLT8_16S:
ALF_EPI_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 | 170012325 | 12,325 |
11 | Total Volume | 170018700 | 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_20202017_EPIGBS | long16S0A3 long16S0A5 | longITS0A3 longITS0A5 |
long16S0B3 long16S0B5 | longITS0B3 longITS0B5 | |
ALF_PLT2_20202017_EPIGBS | long16S0C3 long16S0C5 | longITS0C3 longITS0C5 |
long16S0D3 long16S0D5 | longITS0D3 longITS0D5 | |
ALF_PLT3_20202017_EPIGBS | long16S0E3 long16S0E5 | longITS0E3 longITS0E5 |
long16S0F3 long16S0F5 | longITS0F3 longITS0F5 | |
ALF_PLT4_20202017_EPIGBS | long16S0G3 long16S0G5 | longITS0G3 longITS0G5 |
long16S0H3 long16S0H5 | longITS0H3 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”
...
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 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 (make for 12 plates in 15mL conical tube)
ul/rxn | Reagent | # of rxns | ul needed |
|---|---|---|---|
3 | 5X Kapa HiFi Buffer | 17001300 | 51003900 |
0.45 | 10M dNTPs | 17001300 | 765585 |
0.3 | Kapa HiFi HotStart DNA Pol | 17001300 | 510390 |
7.25 | Ultra Pure H2O | 17001300 | 123259425 |
11 | Total Volume | 1700130018700 | 14,300 |
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_PLT5PLT9_20202017_EPIGBS | long16S0A4 long16S0A7 | longITS0A4 longITS0A7 | |
long16S0B4 long16S0B7 | longITS0B4 longITS0B7 | ||
ALF_PLT6_2020_EPI | long16S0C4 | longITS0C4 | |
long16S0D4 | longITS0D4 | ||
ALF_PLT7_2020_EPI | long16S0E4 | longITS0E4 | |
long16S0F4 | longITS0F4 | ||
ALF_PLT8_2020_EPI | long16S0G4 | longITS0G4 | |
long16S0H4 | longITS0H4PLT10_2017_GBS | long16S0C7 | longITS0C7 |
long16S0D7 | longITS0D7 | ||
ALF_PLT11_2017_GBS | long16S0E7 | longITS0E7 | |
long16S0F7 | longITS0F7 |
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_EPIGBS_PLT5PLT1_16S_3B | ALF_EPIGBS_PLT3_PLT516S_ITS3B | ALF_EPIGBS_PLT6PLT5_16SALF_EPI_PLT6_ITS_3B | ALF_EPIGBS_PLT7_16S_3B | ALF_EPIGBS_PLT9_PLT716S_ITS3B | ALF_EPIGBS_PLT8PLT11_16SALF_EPI_PLT8_ITS_3B |
| NTC | NTC | NTC | |||||||||
BALF_EPI_PLT5_16S | ALF_GBS_EPIPLT1_PLT516S_ITS9E | ALF_EPIGBS_PLT6PLT3_16S_9E | ALF_GBS_EPIPLT5_PLT616S_ITS9E | ALF_EPIGBS_PLT7_16S_9E | ALF_EPIGBS_PLT9_PLT716S_ITS9E | ALF_EPIGBS_PLT8PLT11_16SALF_EPI_PLT8_ITS_9A |
| 0.0002 pM Std | 0.0002 pM Std | 0.0002 pM Std | |||||||||
C | ALF_GBS_EPIPLT1_PLT5ITS_16S3B | ALF_EPIGBS_PLT5PLT3_ITSALF_EPI_PLT6_16S_3B | ALF_EPIGBS_PLT6PLT5_ITS_3B | ALF_EPIGBS_PLT7_16SITS_3B | ALF_EPIGBS_PLT7PLT9_ITSALF_EPI_PLT8_16S_3B | ALF_EPIGBS_PLT8PLT11_ITS_3B |
| 0.002 pM Std | 0.002 pM Std | 0.002 pM Std | |||||||||
D | ALF_GBS_EPIPLT1_PLT5ITS_16S9E | ALF_EPIGBS_PLT5PLT3_ITSALF_EPI_PLT6_16S_9E | ALF_EPIGBS_PLT6PLT5_ITS_9E | ALF_EPIGBS_PLT7_ITS_16S9E | ALF_EPIGBS_PLT7PLT9_ITSALF_EPI_PLT8_16S_9E | ALF_EPIGBS_PLT8PLT11_ITS_9A |
| 0.02 pM Std | 0.02 pM Std | 0.02 pM Std | |||||||||
E | ALF_EPIGBS_PLT5PLT2_16SALF_EPI_PLT5_ITS_3B | ALF_EPIGBS_PLT6PLT4_16S_3B | ALF_EPIGBS_PLT6_ITSALF_EPI_PLT7_16SALF_EPI_PLT7_ITS_3B | ALF_EPIGBS_PLT8_16S_3B | ALF_GBS_EPIPLT10_PLT816S_ITS3B |
| 0.2 pM Std | 0.2 pM Std | 0.2 pM Std | ||||||||||
F | ALF_EPIGBS_PLT5PLT2_16S_9E | ALF_EPIGBS_PLT4_PLT516S_ITS9E | ALF_EPIGBS_PLT6_16SALF_EPI_PLT6_ITS_9E | ALF_EPIGBS_PLT7PLT8_16SALF_EPI_PLT7_ITS_9E | ALF_EPIGBS_PLT8PLT10_16SALF_EPI_PLT8_ITS_9E |
| 2 pM Std | 2 pM Std | 2 pM Std | ||||||||||
G | ALF_EPIGBS_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_ITSPLT2_ITS_3B | ALF_GBS_PLT4_ITS_3B | ALF_GBS_PLT6_ITS_3B | ALF_GBS_PLT8_ITS_3B | ALF_GBS_PLT10_ITS_3B |
| 20 pM Std | 20 pM Std | 20 pM Std | |||
H | ALF_EPIGBS_PLT2_PLT5ITS_16S9E | ALF_EPIGBS_PLT5PLT4_ITSALF_EPI_PLT6_16S_9E | ALF_EPIGBS_PLT6_ITSALF_EPI_PLT7_16S_9E | ALF_EPIGBS_PLT7PLT8_ITSALF_EPI_PLT8_16S_9E | ALF_EPIGBS_PLT8PLT10_ITS_9E |
|
|
|
|
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) | 10060 | 1000600 |
2 ul | Primer Premix (10X) | 10060 | 200120 |
4 ul | Ultra Pure Water | 10060 | 400240 |
16 ul | Total Volume | 10060 | 1600960 |
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
...
...
...
...
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:
...
:
| 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 |
| NTC | NTC | NTC | ||
B | ALF_GBS_PLT1_16S_9E | ALF_GBS_PLT3_16S_9E | ALF_GBS_PLT5_16S_9E | ALF_GBS_PLT7_16S_9E | ALF_GBS_PLT9_16S_9E | ALF_GBS_PLT11_16S_9A |
| 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 |
| 0.002 pM Std | 0.002 pM Std | 0.002 pM Std | ||
D | ALF_GBS_PLT1_ITS_9E | ALF_GBS_PLT3_ITS_9E | ALF_GBS_PLT5_ITS_9E | ALF_GBS_PLT7_ITS_9E | ALF_GBS_PLT9_ITS_9E | ALF_GBS_PLT11_ITS_9A |
| 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 |
| 0.2 pM Std | 0.2 pM Std | 0.2 pM Std | |||
F | ALF_GBS_PLT2_16S_9E | ALF_GBS_PLT4_16S_9E | ALF_GBS_PLT6_16S_9E | ALF_GBS_PLT8_16S_9E | ALF_GBS_PLT10_16S_9E |
| 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 |
| 20 pM Std | 20 pM Std | 20 pM Std | |||
H | ALF_GBS_PLT2_ITS_9E | ALF_GBS_PLT4_ITS_9E | ALF_GBS_PLT6_ITS_9E | ALF_GBS_PLT8_ITS_9E | ALF_GBS_PLT10_ITS_9E |
Results:
Average results for the following plates:
ALF_GBS_PLT1_16S:
ALF_GBS_PLT1_ITS:
ALF_GBS_PLT2_16S:
ALF_GBS_PLT2_ITS:
ALF_GBS_PLT3_16S:
ALF_GBS_PLT3_ITS:
ALF_GBS_PLT4_16S:
ALF_GBS_PLT4_ITS:
ALF_GBS_PLT5_16S:
ALF_GBS_PLT5 ITS:
ALF_GBS_PLT5_16S:
ALF_GBS_PLT5_ITS:
ALF_GBS_PLT6_16S:
ALF_GBS_PLT6_ITS:
ALF_GBS_PLT7_16S:
ALF_GBS_PLT7_ITS:
ALF_GBS_PLT8_16S:
ALF_GBS_PLT8_ITS:
ALF_GBS_PLT9_16S:
ALF_GBS_PLT9_ITS:
ALF_GBS_PLT10_16S:
ALF_GBS_PLT10_ITS:
ALF_GBS_PLT11_16S:
ALF_GBS_PLT11_ITS:
The full result report can be viewed below: