| Info |
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Plates in red have been extracted, PCRed, cleaned, quantified, and normalized by 7-15-22. Bolded red have been qPCRed.6GTL1_16S will need qPCR redone. |
NS6 Plates
6AC1 | 6AC2 | 6AC3 | |
6BM1 | 6BM2 | ||
6LVD1 | 6LVD2 | 6LVD3 | 6LVD4 |
6NW1 | 6NW2 | 6NW3 | 6NW4 |
6RD1 | 6RD2 | 6RD3 | |
6GTL1 | 6GTL2 | ||
MP002 | MP003 | MP006 |
Current total number of plates for ITS/16S PCR: 20 + 1 plate repeat (sm19-fire)
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Make 1:1000 dilutions of column 3 8 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 | NTC | NTC | NTC | B | 0.2TRNL1_TRNL | 2TRNL_16S | 6AC1_16S | 6AC1_ITS | 6AC2_16S | 6AC2_ITS | NTC | NTC | NTC | |||
B | 2TRNL1_TRNL | 2TRNL_16S | 6AC1_16S | 6AC1_ITS | 6AC2_16S | 6AC2_ITS | 0.0002 pM Std | 0.0002 pM Std | 0.0002 pM Std | |||||||
C | 2TRNL1_TRNL | 2TRNL_16S | 6AC1_16S | 6AC1_ITS | 6AC2_16S | 6AC2_ITS | 0.002 pM Std | 0.002 pM Std | 0.002 pM Std | |||||||
D | 0.02 pM Std | 0.02 | 2TRNL1_TRNL | 2TRNL_16S | 6AC1_16S | 6AC1_ITS | 6AC2_16S | 6AC2_ITS | 0.02 pM Std | 0.02 pM Std | 0.02 pM Std | |||||
E | 2TRNL1_TRNL | 2TRNL_16S | 6AC1_16S | 6AC1_ITS | 6AC2_16S | 6AC2_ITS | 0.2 pM Std | 0.2 pM Std | 0.2 pM Std | |||||||
F | 2TRNL1_TRNL | 2TRNL_16S | 6AC1_16S | 6AC1_ITS | 6AC2_16S | 6AC2_ITS | 2 pM Std | 2 pM Std | 2 pM Std | |||||||
G | 20 pM Std | 2TRNL1_TRNL | 2TRNL_16S | 6AC1_16S | 6AC1_ITS | 6AC2_16S | 6AC2_ITS | 20 pM Std | 20 pM Std | 20 pM Std | ||||||
H | 2TRNL1_TRNL | 2TRNL_16S | 6AC1_16S | 6AC1_ITS | 6AC2_16S | 6AC2_ITS |
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Add 16 ul of Illumina Library Quantification MasterMix to each well:
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| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | ||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
A | 2TRNL1_TRNL | 2TRNL_16S | 6AC1_16S | 6AC1_ITS | 6AC2_16S | 6AC2_ITS | NTC | NTC | NTC20 pM Std | 20 pM Std | 20 pM Std | |||
B | 2TRNL1_TRNL | 2TRNL_16S | 6AC1_16S | 6AC1_ITS | 6AC2_16S | 6AC2_ITS | 0.0002 2 pM Std0.0002 | 2 pM Std | 0.0002 2 pM Std | |||||
C | 2TRNL1_TRNL | 2TRNL_16S | 6AC1_16S | 6AC1_ITS | 6AC2_16S | 6AC2_ITS | 0.002 2 pM Std | 0.002 2 pM Std | 0.002 2 pM Std | |||||
D | 2TRNL1_TRNL | 2TRNL_16S | 6AC1_16S | 6AC1_ITS | 6AC2_16S | 6AC2_ITS | 0.02 pM Std | 0.02 pM Std | 0.02 pM Std | |||||
E | 2TRNL1_TRNL | 2TRNL_16S | 6AC1_16S | 6AC1_ITS | 6AC2_16S | 6AC2_ITS | 0.2 002 pM Std | 0.2 002 pM Std | 0.2 002 pM Std | |||||
F | 2TRNL1_TRNL | 2TRNL_16S | 6AC1_16S | 6AC1_ITS | 6AC2_16S | 6AC2_ITS | 2 0.0002 pM Std2 | 0.0002 pM Std | 2 0.0002 pM Std | |||||
G | 2TRNL1_TRNL | 2TRNL_16S | 6AC1_16S | 6AC1_ITS | 6AC2_16S | 6AC2_ITS | 20 pM Std | 20 pM Std | 20 pM StdNTC | NTC | NTC | |||
H | 2TRNL1_TRNL | 2TRNL_16S | 6AC1_16S | 6AC1_ITS | 6AC2_16S | 6AC2_ITS | NTC | NTC | NTC |
Results:
Plate Name | Average Result (nanomoles) |
|---|---|
2TRNL1_TRNL | 10.63 |
2TRNL_16S | 6.02E-01 |
6AC1_16S | 23.35 |
6AC1_ITS | 36.97 |
6AC2_16S | 67.91 |
6AC2_ITS | 28.37 |
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Make 1:1000 dilutions of column 3 8 from the PCR plates by adding 1 ul to 999 ul TE in a deep well plate:
...
Make 1:1000 dilutions of column 3 8 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 |
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| NTC | B |
| 04.0002 pM Std | 0.0002 pM Std | C |
| 0.002 pM Std | 0.002 pM Std | D |
| 0.02 pM Std | 0.02 pM Std | E |
| 0.2 6LVD2_ITS | 6LVD2_16S | 2TRNL3_16S | 2TRNL3_TRNL | 2TRNL2_TRNL | 2TRNL2_16S | 6RD2_16S | 6RD2_ITS | 6GTL1_16S | 2TRNL1_16S_2 |
| NTC |
B | 6LVD2_ITS | 6LVD2_16S | 2TRNL3_16S | 2TRNL3_TRNL | 2TRNL2_TRNL | 2TRNL2_16S | 6RD2_16S | 6RD2_ITS | 6GTL1_16S | 2TRNL1_16S_2 | 04.0002 pM Std | 0.2 0002 pM Std | F |
| 2 pM Std | 2 pM Std | G |
| 20 pM Std | 20 pM Std | H |
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Add 16 ul of Illumina Library Quantification MasterMix to each well:
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ul/rxn
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Reagent
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# of rxns
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ul needed
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10 ul
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KAPA SYBR FAST qPCR MM (2X)
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110
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1100
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2 ul
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Primer Premix (10X)
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110
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220
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4 ul
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Ultra Pure Water
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110
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440
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16 ul
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Total Volume
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110
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1760
Add 4 ul of template, pool, or standards to each well:
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1
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2
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3
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4
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5
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6
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7
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8
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9
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10
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11
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12
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A
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NTC
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NTC
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NTC
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B
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0.0002 pM Std
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0.0002 pM Std
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0.0002 pM Std
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C
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0.002 pM Std
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0.002 pM Std
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0.002 pM Std
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D
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0.02 pM Std
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0.02 pM Std
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0.02 pM Std
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E
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0.2 pM Std
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0.2 pM Std
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0.2 pM Std
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F
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2 pM Std
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2 pM Std
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2 pM Std
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G
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20 pM Std
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20 pM Std
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20 pM Std
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H
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Results:
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Plate Name
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Average Result (nanomoles)
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C | 6LVD2_ITS | 6LVD2_16S | 2TRNL3_16S | 2TRNL3_TRNL | 2TRNL2_TRNL | 2TRNL2_16S | 6RD2_16S | 6RD2_ITS | 6GTL1_16S | 2TRNL1_16S_2 | 0.002 pM Std | 0.002 pM Std |
D | 6LVD2_ITS | 6LVD2_16S | 2TRNL3_16S | 2TRNL3_TRNL | 2TRNL2_TRNL | 2TRNL2_16S | 6RD2_16S | 6RD2_ITS | 6GTL1_16S | 2TRNL1_16S_2 | 0.02 pM Std | 0.02 pM Std |
E | 6LVD2_ITS | 6LVD2_16S | 2TRNL3_16S | 2TRNL3_TRNL | 2TRNL2_TRNL | 2TRNL2_16S | 6RD2_16S | 6RD2_ITS | 6GTL1_16S | 2TRNL1_16S_2 | 0.2 pM Std | 0.2 pM Std |
F | 6LVD2_ITS | 6LVD2_16S | 2TRNL3_16S | 2TRNL3_TRNL | 2TRNL2_TRNL | 2TRNL2_16S | 6RD2_16S | 6RD2_ITS | 6GTL1_16S | 2TRNL1_16S_2 | 2 pM Std | 2 pM Std |
G | 6LVD2_ITS | 6LVD2_16S | 2TRNL3_16S | 2TRNL3_TRNL | 2TRNL2_TRNL | 2TRNL2_16S | 6RD2_16S | 6RD2_ITS | 6GTL1_16S | 2TRNL1_16S_2 | 20 pM Std | 20 pM Std |
H | 6LVD2_ITS | 6LVD2_16S | 2TRNL3_16S | 2TRNL3_TRNL | 2TRNL2_TRNL | 2TRNL2_16S | 6RD2_16S | 6RD2_ITS | 6GTL1_16S | 2TRNL1_16S_2 |
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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) | 110 | 1100 |
2 ul | Primer Premix (10X) | 110 | 220 |
4 ul | Ultra Pure Water | 110 | 440 |
16 ul | Total Volume | 110 | 1760 |
Add 4 ul of template, pool, or standards to each well:
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
A | 6LVD2_ITS | 6LVD2_16S | 2TRNL3_16S | 2TRNL3_TRNL | 2TRNL2_TRNL | 2TRNL2_16S | 6RD2_16S | 6RD2_ITS | 6GTL1_16S | 2TRNL1_16S_2 | 20 pM Std | 20 pM Std |
B | 6LVD2_ITS | 6LVD2_16S | 2TRNL3_16S | 2TRNL3_TRNL | 2TRNL2_TRNL | 2TRNL2_16S | 6RD2_16S | 6RD2_ITS | 6GTL1_16S | 2TRNL1_16S_2 | 2 pM Std | 2 pM Std |
C | 6LVD2_ITS | 6LVD2_16S | 2TRNL3_16S | 2TRNL3_TRNL | 2TRNL2_TRNL | 2TRNL2_16S | 6RD2_16S | 6RD2_ITS | 6GTL1_16S | 2TRNL1_16S_2 | 0.2 pM Std | 0.2 pM Std |
D | 6LVD2_ITS | 6LVD2_16S | 2TRNL3_16S | 2TRNL3_TRNL | 2TRNL2_TRNL | 2TRNL2_16S | 6RD2_16S | 6RD2_ITS | 6GTL1_16S | 2TRNL1_16S_2 | 0.02 pM Std | 0.02 pM Std |
E | 6LVD2_ITS | 6LVD2_16S | 2TRNL3_16S | 2TRNL3_TRNL | 2TRNL2_TRNL | 2TRNL2_16S | 6RD2_16S | 6RD2_ITS | 6GTL1_16S | 2TRNL1_16S_2 | 0.002 pM Std | 0.002 pM Std |
F | 6LVD2_ITS | 6LVD2_16S | 2TRNL3_16S | 2TRNL3_TRNL | 2TRNL2_TRNL | 2TRNL2_16S | 6RD2_16S | 6RD2_ITS | 6GTL1_16S | 2TRNL1_16S_2 | 0.0002 pM Std | 0.0002 pM Std |
G | 6LVD2_ITS | 6LVD2_16S | 2TRNL3_16S | 2TRNL3_TRNL | 2TRNL2_TRNL | 2TRNL2_16S | 6RD2_16S | 6RD2_ITS | 6GTL1_16S | 2TRNL1_16S_2 | NTC | NTC |
H | 6LVD2_ITS | 6LVD2_16S | 2TRNL3_16S | 2TRNL3_TRNL | 2TRNL2_TRNL | 2TRNL2_16S | 6RD2_16S | 6RD2_ITS | 6GTL1_16S | 2TRNL1_16S_2 | NTC | NTC |
Results:
Plate Name | Average Result (nanomoles) |
|---|---|
6LVD2_ITS | 16.09 |
6LVD2_16S | 21.71 |
2TRNL3_16S | 42.69 |
2TRNL3_TRNL | 8.68 |
2TRNL2_TRNL | 7.51 |
2TRNL2_16S | 36.27 |
6RD2_16S | 28.48 |
6RD2_ITS | 29.45 |
6GTL1_16S | 8.2 |
2TRNL1_16S_2 | 32.72 |
Full qPCR results below:
| View file | ||
|---|---|---|
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qPCR NovaSeq6_Set4
Make 1:1000 dilutions of column 3 from the PCR plates by adding 1 ul to 999 ul TE in a deep well plate:
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