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16S0C4 and 16S0D4 were used. |
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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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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).
“ul for pooling” was calculated by multiplying the lowest molarity (3.15) times the maximum V available (35 ul) to get ~110. This result was then divided by each sample’s molarity to obtain the ul needed for equimolar pooling rounded to one decimal place.
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Well Position | Sample | qPCR result | ul for pooling | Repeated qPCR Result | qPCR Duplicated Run Results 02/09 |
1E | SAG192203_R | 54.32 | 2.0 | 1.58 | 2.04 |
2E | SAG_S30P2R_R_2uL | 54.14 | 2.0 | 1.22 | 1.16 |
3E | SAG_S30P2R_R_4uL | 54.03 | 2.0 | 2.05 | 1.34 |
4E | SAG_S30P2R_R_8uL | 11.34 | 9.7 | 5.70E-01 | 3.61E-01 |
5E | Mock_Comm_R | 65.14 | 1.7 | 2.07 | 1.62 |
6E | SAG192199_R_2uL | 71.08 | 1.5 | 2.09 | 1.25 |
7E | SAG192199_R_4uL | 95.41 | 1.2 | 2.34 | 3.91 |
8E | SAG192199_R_8uL | 80.56 | 1.4 | 2.65 | 6.07 |
9E | Blank_ISD_R | 19.23 | 5.7 | 1.8 | 2.25 |
1F | SAG191508_R | 64.52 | 1.7 | 31.69 | 37.26 |
2F | SAG_S38P1R_R_2uL | 60.01 | 1.8 | 1.51 | 1.56 |
3F | SAG_S38P1R_R_4uL | 56.04 | 2.0 | 6.72 | 4F5.75 |
4F | SAG_S38P1R_R_8uL | 3.15 | 34.9 | 3.86E-01 | 1.42E-01 |
5F | Mock_Comm_R | 63.52 | 1.7 | 2.51 | 2.11 |
6F | SAG192077_R_2uL | 65.88 | 1.7 | 2.38 | 1.58 |
7F | SAG192077_R_4uL | 84.27 | 1.3 | 2.37 | 3.1 |
8F | SAG192077_R_8uL | 83.59 | 1.3 | 3.03 | 4.24 |
9F | Blank_ISD_R | 13.12 | 8.4 | 7.78E-01 | 9.15E-01 |
1G | SAG_S16P3R_R | 26.49 | 4.2 | 11.34 | 14.1 |
2G | SAG_S15P4R_R_2uL | 53.51 | 2.1 | 14.31 | 16.26 |
3G | SAG_S15P4R_R_4uL | 26.2 | 4.2 | 1.33 | 1.56 |
4G | SAG_S15P4R_R_8uL | 12.91 | 8.5 | 6.56E-01 | 6.53E-01 |
5G | Mock_Comm_R | 79.7 | 1.4 | 1.9 | 2.14 |
6G | SAG190271_R_2uL | 70.2 | 1.6 | 1.59 | 1.75 |
7G | SAG190271_R_4uL | 62.73 | 1.8 | 1.81 | 1.95 |
8G | SAG190271_R_8uL | 59.32 | 1.9 | 6.33E-01 | 2.61 |
9G | Blank_ISD_R | 20.04 | 5.5 | 6.79E-01 | 1.13 |
1H | SAG_S44P2R_R | 19.92 | 5.5 | 2.4 | 4.46 |
2H | SAG190005_R_2uL | 54.03 | 2.0 | 2.89 | 6.26 |
3H | SAG190005_R_4uL | 57.05 | 1.9 | 2.72 | 3.62 |
4H | SAG190005_R_8uL | 60.28 | 1.8 | 4.37 | 3.46 |
5H | Mock_Comm_R | 64.83 | 1.7 | 2.17 | 2.28 |
6H | SAG190238_R_2uL | 68.19 | 1.6 | 76.62 | 62 |
7H | SAG190238_R_4uL | 55.07 | 2.0 | 2.16 | 1.83 |
8H | SAG190238_R_8uL | 41.35 | 2.7 | 2.5 | 3.88 |
9H | Blank_ISD_R | 18.32 | 6.0 | 5.57E-01 | 1.36 |
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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