After confirming which bead-to-sample ratio I want to use (0.7X, see the bead titration and TapeStation posts for details), I performed bead cleanup and concentration of all samples.
I used KAPA Pure Beads, and followed the protocol and modifications provided in my KAPA bead titration entry.
This iteration of the KAPA Pure Bead protocol was a little more complicated, though, because many of my DNA extractions have slightly different remaining volumes, due to differences in pipet loss, # of gels run, dilutions performed, # of extractions performed for that specimen, and whether samples were included in the titration bead trial. I’m also combining and concentrating the following:
*Extractions 1 and 2 for for specimens 14366, 19054, 52295, 1180630, 50603
+Extraction and titrated bead cleanups for specimens 51732, 51892
Bead cleanups require maintaining an accurate bead-to-sample ratio. However, I want to use all extracted DNA I have for each specimen, to maximize the amount of DNA available for sequencing. This means I couldn’t just take an equal volume from each sample. Instead, I measured the total extraction volume of each sample as I transferred it to a well in my 0.2mL 8X tube strips. I then calculated, for each sample, what bead volume to use to maintain a bead-to-sample ratio of 0.7X.
| 8X tube strip | Catalog # | DNA (uL) | Beads (uL) |
|---|---|---|---|
| A | 14366 * | 90 | 63 |
| A | 19054 * | 100 | 70 |
| A | 52295 * | 100 | 70 |
| A | 1180630 * | 110 | 77 |
| A | 50603 * | 110 | 77 |
| A | 51732 + | 100 | 70 |
| A | 51892 + | 90 | 63 |
| A | |||
| B | 14399 | 55 | 38.5 |
| B | 42137 | 50 | 35 |
| B | 51730 | 70 | 49 |
| B | 51782 | 45 | 31.5 |
| B | 100609 | 45 | 31.5 |
| B | 100610 | 45 | 31.5 |
| B | 1007393 | 45 | 31.5 |
| B | 1018355 | 55 | 38.5 |
| C | 1606824 | 50 | 35 |
| C | 1606826 | 55 | 38.5 |
| C | 1740336 | 55 | 38.5 |
| C | 1740363 | 50 | 35 |
| C | 1740390 | 45 | 31.5 |
| C | 1740407 | 65 | 45.5 |
| C | 50368 | 50 | 35 |
| C | 51727 | 50 | 35 |
| D | 51729 | 45 | 31.5 |
| D | 51857 | 45 | 31.5 |
| D | 51858 | 45 | 31.5 |
| D | 51859 | 45 | 31.5 |
| D | 51860 | 40 | 28 |
| D | 51861 | 30 | 21 |
| D | |||
| D |
After pipetting all samples into 8X tube strips (in the same order as shown in the volume table above), I filled 4 8X tube strips with the corresponding volumes of KAPA Pure Beads (temp-acclimated and vortexed). This allowed me to quickly add the appropriate volume of beads to all samples using a multi-channel pipet.
Eluted all samples in 30uL of Zymo Elution Buffer.