Sam’s Notebook: DNA Isolation and Quantification – C.bairdi Hemocyte Pellets in RNAlater

Isolated DNA from 22 samples (see Qubit spreadsheet in “Results” below for sample IDs) using the Quick DNA/RNA Microprep Kit (ZymoResearch; PDF) according to the manufacturer’s protocol for liquids/cells in RNAlater.

These samples were from an RNA isolation on the following date:

Brief rundown of method:

  • Used 35uL from each RNAlater/hemocyte slurry.
  • Mixed with equal volume of H2O (35uL).
  • Retained DNA on the Zymo-Spin IC-XM columns at 4oC for isolation after RNA isolation.
  • DNA was eluted in 15uL H2O

DNA was quantified on the Roberts Lab Qubit 3.0 using the 1x DNA High Sensitivity Assay (Invitrogen), using 1uL of each sample.

Sam’s Notebook: qPCR – C.bairdi RNA Check for Residual gDNA

Previuosly checked existing crab RNA for residual gDNA on 20200226 and identified samples with yields that were likely too low, as well as samples with residual gDNA. For those samples, was faster/easier to just isolate more RNA and perform the in-column DNase treatment in the ZymoResearch Quick DNA/RNA Microprep Plus Kit; this keeps samples concentrated. So, I isolated more RNA on 20200306 and now need to check for residual gDNA.

Used 2ng of RNA (1uL) in each reaction. A 5uL dilution of each sample was made to a concentration of 2ng/uL. The decision for this quantity was based on the amount of RNA we might use in a reverse transcription reaction (50ng/25uL) and the volume of the resulting cDNA we’d run in a qPCR reaction (1uL). Calculations and the qPCR results (Cq values) are below (Google Sheet).

All reactions were run with 2x SsoFast EVA Green qPCR Master Mix (BioRad) on the Roberts Lab CFX Connect qPCR machine.

All samples were run in duplicate. See the qPCR Report (in Results section below) for plate layout, cycling params, etc.

Master mix calcs are here (Google Sheet):