Two of my 1800s extractions that I want to include in Group 3 are below the concentration needed to get 1000ng in 11uL. I’m going to concentrate them to a lower volume/higher concentration, using the Zymo Genomic DNA Clean and Concentrator Kit (full protocol here).
| Catalog # | Tube | Conc. (ng/uL) | vol. (uL) | desired conc. (ng/uL) | desired vol. (uL) |
|---|---|---|---|---|---|
| 50368 | C7 | 68.3 | 25 | 91 | 18.76, ~19 |
| 42137 | B2 | 46.3 | 26 | 91 | 13.23, ~14 |
desired concentration = 1000ng/11uL = 91ng/uL
desired volume = (conc. + vol.)/desired conc.
Eluted each sample in the desired volume of Zymo EB (heated first to 60C)
Made a 5X dilution of each (1uL DNA + 4uL Zymo EB), then:
Quantified each with the Qubit (1X dsDNA HS protocol and kit)
Standard 1: 34.04
Standard 2: 13568.74
50368: 7.4ng/uL –x5–> 37ng/uL
42137: 5.39ng/ul –x5–> 26.95ng/uLQC’d with Epoch spectrophotometer
260/280 260/230 50368 1.917 2.369 42137 1.9 3.018
Soooo Zymo did not concentrate my samples. I must have lost too mcuh during the clean. I actually cut concentration by a bit. I’ll just have to work with what I got :(
I also may need to fully exclude 42137 due to the high 260/230, indicating possible chemical contamination.