In a pilot experiment I tested three different genomic DNA extraction methods on the non-model organism Pieris mannii (southern small white; Insecta, Lepidoptera, Pieridae). The goal was to generate DNA fragments of ≥30 kbp average length for library preparation for PacBio SMRT long read sequencing (HiFi)).
The three methods/kits used were: Qiagen blood & tissue kit (column assay), Beckman & Coulter DNadvance (magnetic beads kit) and standard Phenol-Chloroform extraction.
Laboratory raised organisms in the prepupal stage (termination of larval growth phase, after last excretion of intestinal content, no chitinous exoskeleton) were harvested and frozen at -20°C. For the DNA extractions the head and the first two leg pairs (ca. 20mg) were cut and processed according to the kit/standard protocols with some modifications for high molecular weight DNA:
- avoidance of freeze-thaw cycles
- pestles were used for grinding the tissue (no liquid nitrogen)
- inverting instead of vortexing
- cell lysis and proteinase K digestion at 56°C and 300rpm overnight
- planned: use of wide-bore pipette tips (impossible because of very long delivery waiting time)
|Method||Avg. DNA fragment length (bp) range||A260/280 range**||A260/230 range**||Nanodrop avg. conc. (ng/µl)||Qubit avg. conc. (ng/µl)|
|Qiagen||1038 - 18366||2.07 - 2.12||1.77 - 2.15||1151.8||104.9|
|Magnetic beads||7350 - 12586||1.98 - 2.11||1.60 - 1.76||1048.7||155.8|
|Phenol-Chloroform||19552 - 27359||2.03 - 2.14||1.94 - 2.10||1389.2||215.4|
*assessed with Agilent Femto Pulse
The DNA fragments seem to be strongly sheared, the samples contaminated with organic compounds (especially the Magnetic bead ones) and the huge difference in concentration between Nanodrop and Qubit measurements may indicate RNA contamination.
How can DNA fragment length and purity be increased? Which are the most important measures? What life stage do you recommend using?
- use of wide-bore pipette tips
- shorten incubation time (cell lysis & proteinase K digestion)
- use freshly eclosed imagines with soft exoskeleton to avoid slimy samples after cell lysis & proteinase K digestion (reason for contaminations?)
- RNase A treatment