DNA and RNA extraction from insects using the ZymoBIOMICS DNA/RNA miniprep kit

Add 750 μl DNA/RNA Shield™ to a sample in a ZR BashingBead Lysis Tube (0.1 & 0.5 mm) and cap tightly. If a sample is already collected in DNA/RNA Shield™, transfer 750 μl liquid sample into a ZR BashingBead Lysis Tube (0.1 & 0.5 mm) and cap tightly.

Perform mechanical homogenization in a ZR BashingBead Lysis Tube (0.1 & 0.5 mm) by securing in a high-speed bead beater fitted with a 2 ml tube holder assembly. Process 4 repetitions of 1 minute bead beating / 3 minutes ice (transfer between bead beater and ice bucket between repetitions).

Centrifuge at 10,000 x g for 30 seconds and transfer up to 400 μl of the supernatant into a nuclease-free tube (not provided in the kit).

Add an equal volume of DNA/RNA Lysis Buffer to the supernatant (1:1) and mix well.

Transfer the sample into a Spin-Away™ Filter (yellow) in a Collection Tube and centrifuge at 10000 x g for 30 seconds. SAVE the flow-through for RNA and the column for DNA purification (resume DNA extraction at step 10)!

Transfer the Spin-Away™ Filter (yellow) into a new Collection Tube and put to one side for processing after RNA extraction is complete.

Add an equal volume of ethanol (95-100%) to flow-through (from step 5 - RNA) and mix well. Example: Add 1.2 ml ethanol to 1.2 ml flow-through. Then transfer the mixture into a Zymo-Spin™ IIICG Column (green) in a Collection Tube and centrifuge. Discard the flow through.

Following RNA binding step, add 400 μl DNA/RNA Wash Buffer to the column, centrifuge at 10,000 x g for 30 seconds and discard the flow-through. Proceed to DNAse I treatment.

For each sample to be treated, prepare DNase I Reaction Mix (5 ul DNase I [1U/ul]; 75 ul Digestion Buffer) in a nuclease-free tube (not provided) and mix by gentle inversion. Then add 80 μl directly into column matrix and incubate at room temperature (20-30°C) for 15 minutes. Proceed with the purification protocol.

Add 400 μl DNA/RNA Prep Buffer to both the DNA column (from step 5) and RNA column (step 9) and centrifuge at 10,000 x g for 30 seconds. Discard the flow-through.

Add 700 μl DNA/RNA Wash Buffer to the column (DNA and RNA) and centrifuge. Discard the flow-through.

Add 400 μl DNA/RNA Wash Buffer to the column (DNA and RNA) and centrifuge at 10,000 x g for 2 minutes to ensure complete removal of the wash buffer. Carefully transfer the column into a nuclease-free tube (not provided).

Add 50-100 μl ZymoBIOMICS™ DNase/RNase-Free Water directly to the column matrix, incubate for 5 minutes, and then centrifuge at 10,000 x g for 30 seconds to elute DNA and RNA from the respective column.

Place Zymo-Spin™ III-HRC Filter in a Collection Tube (one each for DNA and RNA) and add 600 μl ZymoBIOMICS™ HRC Prep Solution. Centrifuge at 8,000 x g for 3 minutes.

Transfer the eluted DNA and RNA (step 13) into a prepared Zymo-Spin™ III-HRC Filter in a nuclease-free tube (not provided). Then centrifuge at exactly 16,000 x g for 3 minutes.