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”
...
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”
...
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 |
| 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_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_9H |
|
|
|
|
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: 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: 8.71 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_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”
...
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”
...
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:
...
...
1
...
2
...
3
...
4
...
5
...
6
...
7
...
8
...
9
...
10
...
11
...
12
...
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_
...
...
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
Results:
Average results for the following plates:
ALF_EPI_PLT1_16S:
ALF_EPI_PLT1_ITS:
ALF_EPI_PLT2_16S:
ALF_EPI_PLT2_ITS: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: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”
...
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”
...
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 |
1300
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_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”
...
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_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:
ul/rxn | Reagent | # of rxns | ul needed |
|---|---|---|---|
10 ul | KAPA SYBR FAST qPCR MM (2X) |
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_16S:PLT2_16S: 14.58 nanomoles
ALF_GBS_PLT1PLT2_ITS: 6.37 nanomoles
ALF_GBS_PLT2PLT3_16S: 18.19 nanomoles
ALF_GBS_PLT2PLT3_ITS: 6.43 nanomoles
ALF_GBS_PLT3PLT4_16S: 6.71 nanomoles
ALF_GBS_PLT3PLT4_ITS: 5.98 nanomoles
ALF_GBS_PLT4PLT5_16S: 7.01 nanomoles
ALF_GBS_PLT4_PLT5 ITS: 5.84 nanomoles
ALF_GBS_PLT5PLT6_16S: : 6.29 nanomoles
ALF_GBS_PLT5 PLT6_ITS: 1.22 nanomoles
ALF_GBS_PLT5PLT7_16S: 1.96 nanomoles
ALF_GBS_PLT5PLT7_ITS: 2.77 nanomoles
ALF_GBS_PLT6PLT8_16S: 9.72 nanomoles
ALF_GBS_PLT6PLT8_ITS: 6.96 nanomoles
ALF_GBS_PLT7PLT9_16S:: 13.38 nanomoles
ALF_GBS_PLT7PLT9_ITS: 15.33 nanomoles
ALF_GBS_PLT8PLT10_16S: 8.09 nanomoles
ALF_GBS_PLT8PLT10_ITS: 8.1 nanomoles
ALF_GBS_PLT9PLT11_16S: 6.30 nanomoles
ALF_GBS_PLT9PLT11_ITS: 7.88 nanomoles
ALF_PLT9_GBS2020_PLT10EPI_16S: 1.69 nanomoles
ALF_PLT9_GBS2020_PLT10EPI_ITS: 0.677 nanomoles
ALF_GBSPLT1_2020_PLT11SOIL_16S: 3.29 nanomoles
ALF_GBSPLT1_2020_PLT11SOIL_ITS: 12.42 nanomoles
The full result report can be viewed below:
| View file | ||
|---|---|---|
|