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.
Use ISD at lower than typical concentrations for all for PCRs, as a positive internal control and recognizing that this will wreck reconstruction of absolute counts. Will add ISD to master mix.
Treat templates with previously high read counts in standard manner, except for lower concentration ISD.
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Sample | Status | Plate | Location | Original Plate Name |
|---|---|---|---|---|
| Low Read | |||
| Low Read | |||
| Low Read | |||
| Low Read | |||
| Low Read | |||
| Low Read | |||
| Low Read | |||
| Low Read | |||
| Avg Read | PA2 | F9 | tube |
| Avg Read | PA1 | A12 | tube |
| Avg Read | PA12 | G3 | AYAYEE_RHIZO_PLATE1 |
| Avg Read | PA12 | F7 | AYAYEE_RHIZO_PLATE1 |
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
A pool 2 µL each |
Tube 127 |
| S43P4RPLT2: E11
|
| S43P4R
|
| S43P4RPLT2: E11
| mock community |
| SAG_S13P3RPLT2: A1
|
| SAG_S13P3R
PLT2: A1 |
|
| blank with ISD only | |||
B pool 2 µL each |
Tube 73 |
| S10P4RPLT1: H2
|
| S10P4R
|
| S10P4RPLT1: H2
| mock community |
| SAG_S3P2RPLT1: B1
|
| SAG_S3P2R
PLT1: B1 |
|
| blank with ISD only | |||
C pool 2 µL each |
PLT1: G3 |
| S14P3RPLT2: E1
|
| S14P3R
|
| S14P3RPLT2: E1
| mock community |
| SAG190267Tube 259
|
| SAG190267
|
| SAG190267
| blank with ISD only | |||
D pool 2 µL each |
PLT1: F7 |
| SAG191507Tube 74
|
| SAG191507
| Tube 74 |
| mock community |
| SAG190430
|
| SAG190430
|
| SAG190430
| blank with ISD only | |||
E pool according to yield |
Tube 127 |
| S43P4RPLT2: E11
|
| S43P4R
|
| S43P4RPLT2: E11
| mock community |
| SAG_S13P3RPLT2: A1
|
| SAG_S13P3R
PLT2: A1 |
|
| blank with ISD only | |||
F pool according to yield |
Tube 73 |
| S10P4RPLT1: H2
|
| S10P4R
|
| S10P4RPLT1: H2
| mock community |
| SAG_S3P2RPLT1: B1
|
| SAG_S3P2R
PLT1: B1 |
|
| blank with ISD only | |||
G pool according to yield |
PLT1: G3 |
| S14P3RPLT2: E1
|
| S14P3R
|
| S14P3R.PLT2: E1
| mock community |
| SAG190267Tube 259
|
| SAG190267
|
| SAG190267
| blank with ISD only | |||
H pool according to yield |
PLT1: F7 |
| SAG191507Tube 74
|
| SAG191507Tube 74
|
| SAG191507
| mock community |
| SAG190430
|
|
| SAG190430Tube 10
| blank with ISD only |
Add 2 ul 1-step primers:
Seal with bubble seals. Vortex briefly. Spin down.
Run on Thermocycler Program GSAF36:
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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:
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