I’ll be using KAPA Pure beads to size-select, clean, and (potentially) concentrate my DNA extractions before library prep. Since I’m not exactly sure what bead ratio will work best to remove <300bp fragments from my samples, I’ll be trying a “titration” trial of several ratios.
For this trial I’ll be using two mid-age samples with high DNA concentrations but a “smeary”, wide range of fragment sizes:
| Catalog Number | Concentration (ng/uL) | Gel size distribution |
|---|---|---|
| USNM 51732 | 89.4 | smeared, 0.3kb |
| USNM 51892 | 86.3 | smeared, 0.15kb |
The KAPA Pure Beads protocol recommends a bead-to-sample ratio of 0.9X - 0.7X to retain fragments >250bp - > 350bp. I’ll test three ratios across this range.
Will be doing cleanup in 0.2mL strip tubes, so will need to assign short labels:
| 0.7X | 0.8X | 0.9X | |
|---|---|---|---|
| USNM 51732 | A.7 | A.8 | A.9 |
| USNM 51892 | B.7 | B.8 | B.9 |
Followed the KAPA Pure Beads protocol (#2, Cleanup of Fragmented DNA in NGS Workflow), with the following notes/modifications:
Mixed fresh aliquot of 80% EtOH using nuclease-free water. It is important to mix a fresh aliquot each time, since EtOH can absorb atmospheric water, slightly changing the concentration over time.
For each rxn, used 5uL NA and diluted to 20uL using Zymo Elution Buffer (5uL DNA + 15uL Zymo Elution Buffer)
With 20uL DNA, the bead volumes for each bead-to-sample ratio are as follows:
0.7X: 20uL DNA + 14uL KAPA Pure Beads
0.8X: 20uL DNA + 16uL KAPA Pure Beads
0.9X 20uL DNA + 18uL KAPA Pure BeadsFirst incubation (step 2.4) for 10 minutes
Dried beads (step 2.13) for 4 minutes
Re-eluted DNA (step 2.15) in 20uL Zymo Elution Buffer. Note that this means the DNA is now diluted by a factor of 4. So my samples, which had concentrations of ~90ng/uL, will now have bead-cleaned concentrations of ~22ng/uL (not accounting for DNA loss during the cleanup process)
Incubated eluting DNA (step 2.16) for 8 minutes