1HILL
Prep
Extract samples using Qiagen Power Soil 96 kit
Quantify via absorbance
Normalize to 10 ng/ul on Nimbus
MasterMix
ul/rxn | Reagent | # of rxns | ul needed |
|---|---|---|---|
3 | 5X Kapa HiFi Buffer | 850 | 2550 |
0.45 | 10M dNTPs | 850 | 382 |
0.3 | Kapa HiFi HotStart DNA Pol | 850 | 255 |
5.25 | HPLC H2O | 850 | 4463 |
9 | Total Volume | 850 | 7650 |
Add 9 ul to each well of a hard shell, full skirt plate. Seal with bubble strips and store in refrigerator until needed.
Array primers into plates with Nimbus
Add 2 ul sample per well
Plate | 16S Primer | ITS Primer |
|---|---|---|
1OR1 |
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1OR2 |
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1OR3 |
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qPCR and Sequencing
16S
Pool concentration = 2.87 nM
1nM Dilution = 69.6 ul Pool + 130.4 ul RSB
Load = 90 ul 1 nM + 9 ul PhiX + 1 ul RSB