| 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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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)
Store in -20 until ready to qPCR.
qPCR
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NovaSeq6_Set1
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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Add 4 ul of template, pool, or standards to each well:
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | ||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
A | NTC | NTC | NTC | B | 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 | |||||||
D0.02 | 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 | |||||||
G20 | 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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Results:
Plate Name | Average Result (nanomoles) |
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Full qPCR results below:
qPCR NovaSeq5_Set2
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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 |
Full qPCR results below:
| View file | ||
|---|---|---|
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qPCR NovaSeq6_Set2
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 |
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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 pM Std | 0.2 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)
Full qPCR results below:
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6AC3_ITS | 6AC3_16S | 6RD1_ITS | 6RD1_16S | 6BM1_ITS | 6BM1_16S | 6LVD1_ITS | 6LVD1_16S | 6GTL1_ITS | 6GTL1_16S | |||
B | 6AC3_ITS | 6AC3_16S | 6RD1_ITS | 6RD1_16S | 6BM1_ITS | 6BM1_16S | 6LVD1_ITS | 6LVD1_16S | 6GTL1_ITS | 6GTL1_16S | ||
C | 6AC3_ITS | 6AC3_16S | 6RD1_ITS | 6RD1_16S | 6BM1_ITS | 6BM1_16S | 6LVD1_ITS | 6LVD1_16S | 6GTL1_ITS | 6GTL1_16S | ||
D | 6AC3_ITS | 6AC3_16S | 6RD1_ITS | 6RD1_16S | 6BM1_ITS | 6BM1_16S | 6LVD1_ITS | 6LVD1_16S | 6GTL1_ITS | 6GTL1_16S | ||
E | 6AC3_ITS | 6AC3_16S | 6RD1_ITS | 6RD1_16S | 6BM1_ITS | 6BM1_16S | 6LVD1_ITS | 6LVD1_16S | 6GTL1_ITS | 6GTL1_16S | ||
F | 6AC3_ITS | 6AC3_16S | 6RD1_ITS | 6RD1_16S | 6BM1_ITS | 6BM1_16S | 6LVD1_ITS | 6LVD1_16S | 6GTL1_ITS | 6GTL1_16S | ||
G | 6AC3_ITS | 6AC3_16S | 6RD1_ITS | 6RD1_16S | 6BM1_ITS | 6BM1_16S | 6LVD1_ITS | 6LVD1_16S | 6GTL1_ITS | 6GTL1_16S | ||
H | 6AC3_ITS | 6AC3_16S | 6RD1_ITS | 6RD1_16S | 6BM1_ITS | 6BM1_16S | 6LVD1_ITS | 6LVD1_16S | 6GTL1_ITS | 6GTL1_16S |
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 | 6AC3_ITS | 6AC3_16S | 6RD1_ITS | 6RD1_16S | 6BM1_ITS | 6BM1_16S | 6LVD1_ITS | 6LVD1_16S | 6GTL1_ITS | 6GTL1_16S | 20 pM Std | 20 pM Std |
B | 6AC3_ITS | 6AC3_16S | 6RD1_ITS | 6RD1_16S | 6BM1_ITS | 6BM1_16S | 6LVD1_ITS | 6LVD1_16S | 6GTL1_ITS | 6GTL1_16S | 2 pM Std | 2 pM Std |
C | 6AC3_ITS | 6AC3_16S | 6RD1_ITS | 6RD1_16S | 6BM1_ITS | 6BM1_16S | 6LVD1_ITS | 6LVD1_16S | 6GTL1_ITS | 6GTL1_16S | 0.2 pM Std | 0.2 pM Std |
D | 6AC3_ITS | 6AC3_16S | 6RD1_ITS | 6RD1_16S | 6BM1_ITS | 6BM1_16S | 6LVD1_ITS | 6LVD1_16S | 6GTL1_ITS | 6GTL1_16S | 0.02 pM Std | 0.02 pM Std |
E | 6AC3_ITS | 6AC3_16S | 6RD1_ITS | 6RD1_16S | 6BM1_ITS | 6BM1_16S | 6LVD1_ITS | 6LVD1_16S | 6GTL1_ITS | 6GTL1_16S | 0.002 pM Std | 0.002 pM Std |
F | 6AC3_ITS | 6AC3_16S | 6RD1_ITS | 6RD1_16S | 6BM1_ITS | 6BM1_16S | 6LVD1_ITS | 6LVD1_16S | 6GTL1_ITS | 6GTL1_16S | 0.0002 pM Std | 0.0002 pM Std |
G | 6AC3_ITS | 6AC3_16S | 6RD1_ITS | 6RD1_16S | 6BM1_ITS | 6BM1_16S | 6LVD1_ITS | 6LVD1_16S | 6GTL1_ITS | 6GTL1_16S | NTC | NTC |
H | 6AC3_ITS | 6AC3_16S | 6RD1_ITS | 6RD1_16S | 6BM1_ITS | 6BM1_16S | 6LVD1_ITS | 6LVD1_16S | 6GTL1_ITS | 6GTL1_16S | NTC | NTC |
Results:
Plate Name | Average Result (nanomoles) |
|---|---|
6AC3_ITS | 4.125 |
6AC3_16S | 7.46 |
6RD1_ITS | 10.152 |
6RD1_16S | 15.709 |
6BM1_ITS | 7.874 |
6BM1_16S | 4.842 |
6LVD1_ITS | 4.532 |
6LVD1_16S | 8.102 |
6GTL1_ITS | 5.992 |
6GTL1_16S | Needs to be redone |
Full qPCR results below:
| View file | ||
|---|---|---|
|
qPCR NovaSeq6_Set3
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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Plate Name | Average Result (nanomoles) |
|---|---|
Full qPCR results below:
qPCR
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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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Plate Name | Average Result (nanomoles) |
|---|---|
Full qPCR results below:
qPCR
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NovaSeq6_Set5
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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Plate Name | Average Result (nanomoles) |
|---|---|
Full qPCR results below:
qPCR
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NovaSeq6_Set6
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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