...
4AH1 | 4AH2 | ||||
4PA1 | 4PA2 | 4PA3 | 4PA4 | 4PA5 | 4PA6 |
4MR1 | 4MR2 | ||||
4SC1 | 4SC2 | ||||
4LVD1 | 4LVD2 | 4LVD3 | |||
4FB1 | 4FB2 | 4FB3 |
MasterMix (make for 16 plates in 50mL conical tube)
...
Average results for the following plates (column 3):
4PA3_16S: 4.04 nanomoles
4PA3_ITS: 46.07 nanomoles
4PA4_16S: 71.0 nanomoles
4PA4_ITS: 68.5 nanomoles
4PA5_16S: 10.26 nanomoles
4PA5_ITS: 41.22 nanomoles
4PA6_16S: 11.57 nanomoles
4PA6_ITS: 44.52 nanomoles
Quantities look good for sequencing. The full result report can be viewed below:
| View file | ||
|---|---|---|
|
MasterMix (make for 12 plates in 15mL conical tube)
...
Average results for the following plates (column 3):
4MR1_16S: 92.23 nanomoles
4MR24MR1_ITS: 53.47 nanomoles
4SC14MR2_16S: 105.34 nanomoles
4MR2_ITS: 66.71 nanomoles
4SC1_16S: 46.64 nanomoles
4SC1_ITS: 47.77 nanomoles
4SC2_16S: 65.97 nanomoles
4SC2_ITS: 55.69 nanomoles
Quantities look good for sequencing. The full result report can be viewed below:
| View file | ||
|---|---|---|
|
MasterMix (make for
...
24 plates in 50mL conical tube)
ul/rxn | Reagent | # of rxns | ul needed |
|---|---|---|---|
3 | 5X Kapa HiFi Buffer | 10002500 | 30007500 |
0.45 | 10M dNTPs | 10002500 | 4501125 |
0.3 | Kapa HiFi HotStart DNA Pol | 10002500 | 300750 |
7.25 | HPLC H2O | 10002500 | 725018125 |
11 | Total Volume | 10002500 | 1100027500 |
Add 11 ul to each well of a hard shell, full skirt plate. Seal with bubble strips and store in refrigerator until needed label “NovaSeq4 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 NovaSeq4_
...
Plate4
Make 1:1000 dilutions of column 3 from the PCR plates by adding 1 ul to 999 ul TE in a deep well plate:
...
Average results for the following plates (column 3):
4LVD1_16S: 112.54 nanomoles
4LVD1_ITS: 24.71 nanomoles
4LVD2_16S: 95.57 nanomoles
4LVD2_ITS: 34.28 nanomoles
4LVD3_16S: 112.47 nanomoles
4LVD3_ITS: 101.03 nanomoles
Quantities look good for sequencing. The full result report can be viewed below:
| View file | ||
|---|---|---|
|
qPCR NovaSeq4_Plate5
Make 1:1000 dilutions of column 3 from the PCR plates by adding 1 ul to 999 ul TE in a deep well plate:
Use other half of NS4_Plate 4 dilution plate to do NS4_Plate5 dilution plate.
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
A | ______ | ______ | ______ | ______ | ______ | ______ | 4FB1_16S | 4FB1_ITS | 4FB2_16S | 4FB2_ITS | 4FB3_16S | 4FB3_ITS |
B | ______ | ______ | ______ | ______ | ______ | ______ | 4FB1_16S | 4FB1_ITS | 4FB2_16S | 4FB2_ITS | 4FB3_16S | 4FB3_ITS |
C | ______ | ______ | ______ | ______ | ______ | ______ | 4FB1_16S | 4FB1_ITS | 4FB2_16S | 4FB2_ITS | 4FB3_16S | 4FB3_ITS |
D | ______ | ______ | ______ | ______ | ______ | ______ | 4FB1_16S | 4FB1_ITS | 4FB2_16S | 4FB2_ITS | 4FB3_16S | 4FB3_ITS |
E | ______ | ______ | ______ | ______ | ______ | ______ | 4FB1_16S | 4FB1_ITS | 4FB2_16S | 4FB2_ITS | 4FB3_16S | 4FB3_ITS |
F | ______ | ______ | ______ | ______ | ______ | ______ | 4FB1_16S | 4FB1_ITS | 4FB2_16S | 4FB2_ITS | 4FB3_16S | 4FB3_ITS |
G | ______ | ______ | ______ | ______ | ______ | ______ | 4FB1_16S | 4FB1_ITS | 4FB2_16S | 4FB2_ITS | 4FB3_16S | 4FB3_ITS |
H | ______ | ______ | ______ | ______ | ______ | ______ | 4FB1_16S | 4FB1_ITS | 4FB2_16S | 4FB2_ITS | 4FB3_16S | 4FB3_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) | 80 | 800 |
2 ul | Primer Premix (10X) | 80 | 160 |
4 ul | Ultra Pure Water | 80 | 320 |
16 ul | Total Volume | 80 | 1280 |
Add 4 ul of template, pool, or standards to each well:
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
A | 4FB1_16S | 4FB1_ITS | 4FB2_16S | 4FB2_ITS | 4FB3_16S | 4FB3_ITS |
| NTC | NTC | NTC | ||
B | 4FB1_16S | 4FB1_ITS | 4FB2_16S | 4FB2_ITS | 4FB3_16S | 4FB3_ITS |
| 0.0002 pM Std | 0.0002 pM Std | 0.0002 pM Std | ||
C | 4FB1_16S | 4FB1_ITS | 4FB2_16S | 4FB2_ITS | 4FB3_16S | 4FB3_ITS |
| 0.002 pM Std | 0.002 pM Std | 0.002 pM Std | ||
D | 4FB1_16S | 4FB1_ITS | 4FB2_16S | 4FB2_ITS | 4FB3_16S | 4FB3_ITS |
| 0.02 pM Std | 0.02 pM Std | 0.02 pM Std | ||
E | 4FB1_16S | 4FB1_ITS | 4FB2_16S | 4FB2_ITS | 4FB3_16S | 4FB3_ITS |
| 0.2 pM Std | 0.2 pM Std | 0.2 pM Std | ||
F | 4FB1_16S | 4FB1_ITS | 4FB2_16S | 4FB2_ITS | 4FB3_16S | 4FB3_ITS |
| 2 pM Std | 2 pM Std | 2 pM Std | ||
G | 4FB1_16S | 4FB1_ITS | 4FB2_16S | 4FB2_ITS | 4FB3_16S | 4FB3_ITS |
| 20 pM Std | 20 pM Std | 20 pM Std | ||
H | 4FB1_16S | 4FB1_ITS | 4FB2_16S | 4FB2_ITS | 4FB3_16S | 4FB3_ITS |
|
|
|
|
Results:
Average results for the following plates (column 3):
4FB1_16S: 28.57 nanomoles
4FB1_ITS: 45.42 nanomoles
4FB2_16S: 70.82 nanomoles
4FB2_ITS: 71.52 nanomoles
4FB3_16S: 73.04 nanomoles
4FB3_ITS: 72.32 nanomoles
Quantities look good for sequencing. The full result report can be viewed below:
| View file | ||
|---|---|---|
|