...
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
A | ALF_PLT5_TN_Col116S_Col3 | ALF_PLT5_TN_Col516S_Col9 | ALF_PLT5_TN_ITS_Col7Col3 | ALF_PLT5_TN_Col11ITS_Col9 | ALF_PLT12_TN_16S_Col1Col3 | ALF_PLT12_TN_16S_Col5Col9 | ALF_PLT12_TN_ITS_Col7Col3 | ALF_PLT12_TN_Col11ITS_Col9 |
|
|
| NTC |
B | ALF_PLT5_TN_Col116S_Col3 | ALF_PLT5_TN_Col516S_Col9 | ALF_PLT5_TN_ITS_Col7Col3 | ALF_PLT5_TN_ITS_Col11Col9 | ALF_PLT12_TN_16S_Col1Col3 | ALF_PLT12_TN_16S_Col5Col9 | ALF_PLT12_TN_Col7ITS_Col3 | ALF_PLT12_TN_ITS_Col11Col9 |
|
| 04.0002 pM Std | 0.0002 pM Std |
C | ALF_PLT5_TN_16S_Col1Col3 | ALF_PLT5_TN_16S_Col5Col9 | ALF_PLT5_TN_ITS_Col7Col3 | ALF_PLT5_TN_ITS_Col11Col9 | ALF_PLT12_TN_Col116S_Col3 | ALF_PLT12_TN_16S_Col5Col9 | ALF_PLT12_TN_Col7ITS_Col3 | ALF_PLT12_TN_ITS_Col11Col9 |
|
| 0.002 pM Std | 0.002 pM Std |
D | ALF_PLT5_TN_16S_Col1Col3 | ALF_PLT5_TN_16S_Col5Col9 | ALF_PLT5_TN_Col7ITS_Col3 | ALF_PLT5_TN_ITS_Col11Col9 | ALF_PLT12_TN_Col116S_Col3 | ALF_PLT12_TN_16S_Col5Col9 | ALF_PLT12_TN_ITS_Col7Col3 | ALF_PLT12_TN_ITS_Col11Col9 |
|
| 0.02 pM Std | 0.02 pM Std |
E | ALF_PLT5_TN_Col116S_Col3 | ALF_PLT5_TN_Col516S_Col9 | ALF_PLT5_TN_Col7ITS_Col3 | ALF_PLT5_TN_ITS_Col11Col9 | ALF_PLT12_TN_16S_Col1Col3 | ALF_PLT12_TN_16S_Col5Col9 | ALF_PLT12_TN_ITS_Col7Col3 | ALF_PLT12_TN_Col11ITS_Col9 |
|
| 0.2 pM Std | 0.2 pM Std |
F | ALF_PLT5_TN_16S_Col1Col3 | ALF_PLT5_TN_16S_Col5Col9 | ALF_PLT5_TN_ITS_Col7Col3 | ALF_PLT5_TN_ITS_Col11Col9 | ALF_PLT12_TN_16S_Col1Col3 | ALF_PLT12_TN_Col516S_Col9 | ALF_PLT12_TN_ITS_Col7Col3 | ALF_PLT12_TN_Col11ITS_Col9 |
|
| 2 pM Std | 2 pM Std |
G | ALF_PLT5_TN_16S_Col1Col3 | ALF_PLT5_TN_16S_Col5Col9 | ALF_PLT5_TN_ITS_Col7Col3 | ALF_PLT5_TN_Col11ITS_Col9 | ALF_PLT12_TN_16S_Col1Col3 | ALF_PLT12_TN_Col516S_Col9 | ALF_PLT12_TN_ITS_Col7Col3 | ALF_PLT12_TN_ITS_Col11Col9 |
|
| 20 pM Std | 20 pM Std |
H | ALF_PLT5_TN_16S_Col1Col3 | ALF_PLT5_TN_Col516S_Col9 | ALF_PLT5_TN_Col7ITS_Col3 | ALF_PLT5_TN_Col11ITS_Col9 | ALF_PLT12_TN_16S_Col1Col3 | ALF_PLT12_TN_16S_Col5Col9 | ALF_PLT12_TN_ITS_Col7Col3 | ALF_PLT12_TN_ITS_Col11Col9 |
|
|
|
|
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) | 6070 | 600700 |
2 ul | Primer Premix (10X) | 6070 | 120140 |
4 ul | Ultra Pure Water | 6070 | 240280 |
16 ul | Total Volume | 6070 | 9601120 |
Add 4 ul of template, pool, or standards to each well:
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| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
A | ALF_PLT5_TN_Col1ALF_PLT5_TN_Col516S_Col3 | ALF_PLT5_TN_Col7ALF_PLT5_TN_Col11ITS_Col3 | ALF_PLT12_TN_16S_Col1Col3 | ALF_PLT12_TN_ITS_Col5Col3 | ALF_PLT5_PLT1212_TN16S_Col7POOLALF_PLT12_TN_Col11 | HPAU_PP |
| NTC | NTC | NTC | ||
B | ALF_PLT5_TN_Col1ALF_PLT5_TN_Col516S_Col3 | ALF_PLT5_TN_Col7ALF_PLT5_TN_Col11ITS_Col3 | ALF_PLT12_TN_Col116S_Col3 | ALF_PLT12_TN_ITS_Col5Col3 | ALF_PLT5_PLT1212_TN16S_Col7POOLALF_PLT12_TN_Col11 | HPAU_PP |
| 0.0002 pM Std | 0.0002 pM Std | 0.0002 pM Std | ||
C | ALF_PLT5_TN_Col116S_Col3 | ALF_PLT5_TN_ITS_Col5Col3 | ALF_PLT5PLT12_TN_Col7ALF_PLT5_TN_Col1116S_Col3 | ALF_PLT12_TN_Col1ITS_Col3 | ALF_PLT12PLT5_TN12_Col5ALF16S_PLT12POOL_TN_Col710000ALF | RHIR_PLT12PP_TN_Col11LN2 |
| 0.002 pM Std | 0.002 pM Std | 0.002 pM Std | ||
D | ALF_PLT5_TN_Col116S_Col3 | ALF_PLT5_TN_Col5ITS_Col3 | ALF_PLT5PLT12_TN_Col7ALF_PLT5_TN_Col1116S_Col3 | ALF_PLT12_TN_ITS_Col1Col3 | ALF_PLT12PLT5_TN12_Col5ALF16S_PLT12POOL_TN_Col710000ALF | RHIR_PLT12PP_TN_Col11LN2 |
| 0.02 pM Std | 0.02 pM Std | 0.02 pM Std | ||
E | ALF_PLT5_TN_Col1ALF_PLT5_TN_Col516S_Col3 | ALF_PLT5_TN_Col7ALF_PLT5_TN_Col11ITS_Col3 | ALF_PLT12_TN_16S_Col1Col3 | ALF_PLT12_TN_ITS_Col5Col3 | ALF_PLT12PLT5_12_TNITS_Col7POOLALF | RHIR_PLT12PP_TN_Col11LN3 |
| 0.2 pM Std | 0.2 pM Std | 0.2 pM Std | ||
F | ALF_PLT5_TN_Col1ALF_PLT5_TN_Col516S_Col3 | ALF_PLT5_TN_Col7ALF_PLT5_TN_Col11ITS_Col3 | ALF_PLT12_TN_16S_Col1Col3 | ALF_PLT12_TN_ITS_Col5Col3 | ALF_PLT5_PLT1212_TNITS_Col7POOLALF | RHIR_PLT12PP_TN_Col11LN3 |
| 2 pM Std | 2 pM Std | 2 pM Std | ||
G | ALF_PLT5_TN_Col1ALF_PLT5_TN_Col516S_Col3 | ALF_PLT5_TN_Col7ALF_PLT5_TN_Col11ITS_Col3 | ALF_PLT12_TN_Col116S_Col3 | ALF_PLT12_TN_ITS_Col5Col3 | ALF_PLT12PLT5_TN12_Col7ALFITS_PLT12POOL_TN_Col1110000 |
| 20 pM Std | 20 pM Std | 20 pM Std | |||
H | ALF_PLT5_TN_Col1ALF_PLT5_TN_Col516S_Col3 | ALF_PLT5_TN_Col7ALF_PLT5_TN_Col11ITS_Col3 | ALF_PLT12_TN_Col116S_Col3 | ALF_PLT12_TN_Col5ITS_Col3 | ALF_PLT12PLT5_TN12_Col7ALFITS_PLT12POOL_TN_Col1110000 |
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Results:
Average results are in nanomoles below:
ALF_PLT5_TN_16S_Col1Col3: 18.49
ALF_PLT5_TN_ITS_Col5Col3: 35.83
ALF_PLT5PLT12_TN_16S_Col7Col3: 25.41
ALF_PLT5PLT12_TN_ITS_Col11Col3: 36
ALF_PLT12PLT5_12_TN16S_Col1POOL: 40.97
ALF_PLT5_12_PLT1216S_TNPOOL_Col510000: .0281
ALF_PLT12PLT5_12_TNITS_Col7POOL: 64.71
ALF_PLT12_TN_Col11:
...
_PLT5_12_ITS_POOL_10000: 7.64
HPAU_PP: 5.84
RHIR_PP_LN2: 22.25
RHIR_PP_LN3: 18.03
iSeq Run 16S POOL ONLY:
Dilute to 1 nM based off qPCR results. qPCR results are in pM, but 1:1000 dilution used. The results are effectively in nM for pool.
1001000/Results = ul of Pool to Add
100/____ = ____ 1000/41= 24.4 uL of Pool to Add
100 1000 - uL of Pool to Add = ul of “10 mM Tris 8.5” to Add
100- ____ = ____1000- 24.4= 975.6 uL of 10mM Tris 8.5
Dilute 1 nM full pool to loading concentration of 50 pM:
Add 5 6 ul 1 nM Pool to 85 82 ul “10 mM Tris 8.5” and 10 12 ul 50 pM PhiX
Remove iSeq 100 i1 Flow Cell from refrigerator 5’s crisper drawer and open white foil pack and allow to equilibrate to RT for 10-15 minutes.
Open “iSeq 100 i1 Reagent Cartridge v2”. Turn on iSeq100
Click on “Sequence”. Watch Video. Do what video tells you to do. Follow on screen instructions until run starts.