Will clean and size-select my 1800s re-extractions to remove the shortest fragments. Followed the KAPA Pure Beads protocol (#2, Cleanup of Fragmented DNA in NGS Workflow).
Notes:
Mixed fresh 8-% EtOH. Need 400uL*5 samples = 2000uL, so miz 2200uL 80% EtOH -> 1760uL EtOH + 440uL nuc-free H2O.
Currently have 49uL of each sample (eluted Zymo in 50uL each, used 1uL for Qubit)
Want 0.7x bead-to-sample ratio (should remove <350bp). Bead volume is 0.7*49uL = 34.3uL KAPA Pure beads to each sample (50368, 50603, 19054, 14399, 42137)
Incubated on beads (step 2.4) for 15min to maximize DNA retention
Eluted in 20uL to help concentrate (eluted in Zymo EB)
Qubit of cleaned DNA
Based on post-Zymo qubits, expect 50368 and 42137 may have 20uL concentrations above the threshold of 100ng/uL (past which Qubit cannot accurately quantify), wo will dilute to 1/2 concentration (2uL sample + 2uL Zymo EB)
Performed Qubit quantification as normal (protocol found here).
Standard #1: 37.63
Standard #2: 16705.55
50368 (dil): 67.6 ng/uL –x2–> 135.2 ng/uL
50603: 10.3 ng/uL
19054: 0.520 ng/uL
14399: 42.8 ng/uL
42137 (dil): 27.2 ng/uL –x2–> 54.4 ng/uL