Gregg Randolph Linda van Diepen Alex Buerkle
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Pull aliquots of some high read count and low read count samples. (Pending new low read samples).
Quantify all samples not including blanks or mock community.
For previously low yielding samples, repeat at typical template amount, and at 2x, and 4x volume added to the reaction (in column 2,3,4, and 6, 7, 8). 6x8 samples in low yield, 8 in high yield, 8 of mock community, 8 of blank (ISD only). 72 reactions with different templates. See template below for clarification.
Template layout is replicated/duplicated, with different barcodes for duplicates.
1 set of 36 products will not be adjusted for pooling.
1 set of 36 will be adjusted for pooling per qPCR results.
Start Reaction Plate Setup:
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Make 1:1000 dilutions of all samples from the PCR plates by adding 1 ul to 999 ul TE in a deep well plate:
Add 4 ul of template, pool, or standards to each well:
1
2
3
4
5
6
7
8
9
10
11
12
A
SAG192203
Tube 127
mock community
blank with ISD only
NTC
NTC
NTC
B
SAG191508
Tube 73
mock community
blank with ISD only
0.0002 pM Std
0.0002 pM Std
0.0002 pM Std
C
SAG_S16P3R
PLT1: G3
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | ||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
A |
Tube 127 | mock community | blank with ISD only |
|
| NTC | B |
Tube 73 | mock community | blank with ISD only |
| 04.0002 pM Std | 0.0002 pM Std | C |
PLT1: G3 | mock community | blank with ISD only |
| 0.002 pM Std | 0.002 pM Std | D |
PLT1: F7 | mock community | blank PLT2: G6 |
PLT2: G6 |
PLT2: G6 | mock community |
Tube 123 |
Tube 123 |
Tube 123 | blank with ISD only |
|
| NTC |
B |
Tube 73 |
PLT2: B9 |
PLT2: B9 |
PLT2: B9 | mock community |
Tube 96 |
Tube 96 |
Tube 96 | blank with ISD only |
| 004.02 0002 pM Std | 0.02 0002 pM Std | ||||||||||||||||||||||
EC |
Tube 127 | mock community | blank
PLT1: G3 |
PLT2: B2 |
PLT2: B2 |
PLT2: B2 | mock community |
Tube 258 |
Tube 258 |
Tube 258 | blank with ISD only |
| 0.2 002 pM Std | 0.2 002 pM Std | ||||||||||||||||||||
FD |
Tube 73 | mock community | blank
PLT1: F7 |
Tube 33 |
Tube 33 |
Tube 33 | mock community |
Tube 255 |
Tube 255 |
Tube 255 | blank with ISD only |
| 2 0.02 pM Std | 2 0.02 pM Std | G | |||||||||||||||||||
E |
Tube 127 |
PLT1: G3 | mock community | blank PLT2: G6 |
PLT2: G6 |
PLT2: G6 | mock community |
Tube 123 |
Tube 123 |
Tube 123 | blank with ISD only |
| 20 0.2 pM Std | 20 0.2 pM Std | H | |||||||||||||||||||
F |
Tube 73 |
PLT1: F7 | mock community | blank PLT2: B9 |
PLT2: B9 |
PLT2: B9 | mock community |
Tube 96 |
Tube 96 |
Tube 96 | blank with ISD only |
|
|
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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)
...
100
...
1000
...
2 ul
...
Primer Premix (10X)
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100
...
200
...
4 ul
...
Ultra Pure Water
...
100
...
400
...
16 ul
...
Total Volume
...
100
...
1600
2 pM Std | 2 pM Std | |||||||||||
G |
PLT1: G3 |
PLT2: B2 |
PLT2: B2 |
PLT2: B2 | mock community |
Tube 258 |
Tube 258 |
Tube 258 | blank with ISD only |
| 20 pM Std | 20 pM Std |
H |
PLT1: F7 |
Tube 33 |
Tube 33 |
Tube 33 | mock community |
Tube 255 |
Tube 255 |
Tube 255 | blank with ISD only |
|
|
|
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) | 100 | 1000 |
2 ul | Primer Premix (10X) | 100 | 200 |
4 ul | Ultra Pure Water | 100 | 400 |
16 ul | Total Volume | 100 | 1600 |
Add 4 ul of template, pool, or standards to each well:
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
A |
Tube 127 |
PLT2: G6 |
PLT2: G6 |
PLT2: G6 | mock community |
Tube 123 |
Tube 123 |
Tube 123 | blank with ISD only | NTC | NTC | NTC | ||||||||
B |
Tube 73 |
PLT2: B9 |
PLT2: B9 |
PLT2: B9 | mock community |
Tube 96 |
Tube 96 |
Tube 96 | blank with ISD only | 0.0002 pM Std | 0.0002 pM Std | 0.0002 pM Std | ||||||||
C |
PLT1: G3 |
PLT2: B2 |
PLT2: B2 |
PLT2: B2 | mock community |
Tube 258 |
Tube 258 |
Tube 258 | blank with ISD only | 0.002 pM Std | 0.002 pM Std | 0.002 pM Std | ||||||||
D |
PLT1: F7 |
Tube 33 |
Tube 33 |
Tube 33 | mock community |
Tube 255 |
Tube 255 |
Tube 255 | blank with ISD only | 0.002 02 pM Std | 0.002 02 pM Std | 0.002 pM Std | D |
PLT1: F7 | mock community | blank 02 pM Std | ||||
E |
Tube 127 |
PLT2: G6 |
PLT2: G6 |
PLT2: G6 | mock community |
Tube 123 |
Tube 123 |
Tube 123 | blank with ISD only | 0.02 2 pM Std | 0.02 2 pM Std | 0.02 2 pM Std | ||||||||
EF |
Tube 127 | mock community | blank with ISD only | 0.2 pM Std | 0.2 pM Std | 0.2 pM Std | F |
Tube 73 | mock community73 |
PLT2: B9 |
PLT2: B9 |
PLT2: B9 | mock community |
Tube 96 |
Tube 96 |
Tube 96 | blank with ISD only | 2 pM Std | 2 pM Std | 2 pM Std |
G |
PLT1: G3 |
PLT2: B2 |
PLT2: B2 |
PLT1PLT2: G3B2 | mock community |
Tube 258 |
Tube 258 |
Tube 258 | blank with ISD only | 20 pM Std | 20 pM Std | 20 pM Std | ||||||||
H |
PLT1: F7 |
Tube 33 |
Tube 33 |
Tube 33 | mock community |
Tube 255 |
Tube 255 |
Tube 255 | blank with ISD only |
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Results:
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Red samples (bottom half) will either just be pooled per qPCR numbers or concentrated via SpeedVac, reconstituted to a higher concentration, and pooled by qPCR results. (Pending method instructions).
qPCR blue/red pools:
Make 1:1000 dilutions of blue and red pools by adding 1 ul to 999 ul TE in a 1.5mL tubes.
Run each pool in triplicate.
Add 16 ul of Illumina Library Quantification MasterMix to each well:
...