DNA/RNA extraction

Why it matters

Almost every molecular method in this course starts with clean nucleic acid. PCR, qPCR, sequencing and cloning all fail, or quietly give wrong answers, when the DNA or RNA going in is degraded, contaminated or too dilute. A biological technician may run dozens of extractions a day, and a medical laboratory technologist may run them on automated platforms for infectious disease or genetic tests. In every case the job is the same: get the nucleic acid out of the cells, away from everything else, and prove it is good enough to use.

The core idea: lyse, bind, wash, elute

Most modern extractions use a silica spin column or magnetic silica beads. The steps are the same in almost every kit:

1. Lyse. Break open cells and nuclei with a lysis buffer (detergent plus salts), often with an enzyme such as proteinase K to digest proteins. Tough samples (plant tissue, bacteria with thick walls) may need mechanical disruption too.
2. Bind. Add a high concentration of a chaotropic salt (and usually ethanol). Under these conditions nucleic acids stick to silica, while most proteins and other debris do not.
3. Wash. Spin wash buffers (usually ethanol-based) through the column to remove salts and leftover contaminants. A final "dry spin" removes residual ethanol, which would inhibit later reactions.
4. Elute. Add a small volume of low-salt buffer or nuclease-free water. Nucleic acid releases from the silica and collects in a clean tube.

Older methods, such as phenol-chloroform extraction (and its RNA cousin, TRIzol), separate nucleic acids by partitioning into a water layer. They work well but use hazardous solvents, so they are done only in a fume hood after specific training.

DNA versus RNA

RNA is far less forgiving than DNA:

• RNases are everywhere (skin, dust, many reagents) and do not need cofactors. RNA work uses RNase-free tubes and tips, fresh gloves, a clean bench and often a dedicated area.
• Work fast and cold. Samples are kept on ice or put into a stabilising reagent immediately.
• Remove DNA. RNA preps for gene expression are usually treated with DNase so genomic DNA does not show up in later qPCR.

Checking what you got

You always measure yield and purity before using an extract, usually on a microvolume UV spectrophotometer:

Worked example. A DNA eluate (undiluted, 1 cm path equivalent) reads A260 = 0.84, A280 = 0.46, A230 = 0.41.

• Concentration = 0.84 x 50 ng/uL = 42 ng/uL.
• A260/A280 = 0.84 / 0.46 = 1.83: little protein.
• A260/A230 = 0.84 / 0.41 = 2.05: little salt carryover.

This extract looks usable. If A260/A230 had been 0.9, you would suspect chaotropic salt carryover, often from a skipped dry spin or wash, and expect PCR to struggle.

For RNA, purity ratios are not enough: integrity matters too. Labs check it on an automated electrophoresis instrument that gives an RNA integrity number (RIN) from 1 (degraded) to 10 (intact).

Following a protocol

A kit protocol is a controlled procedure, not a suggestion. Before you start, read it once end to end, and mark:

• Steps you must prepare in advance, such as adding ethanol to a wash buffer concentrate or pre-heating a block.
• Steps that are easy to get wrong, such as which wash buffer goes first or whether to discard the flow-through.
• Stopping points, where the sample can be safely stored.
• Your controls: an extraction blank (no sample) shows whether reagents or technique introduced contamination.

Record lot numbers, the protocol version and any deviation in your notebook, as you learned in the lab data course.

Safety and real-world training

Real samples can be infectious (clinical specimens, cultured pathogens) and lysis reagents are often irritant or toxic. Extraction is done only in a teaching or employer lab, at the right biosafety level, after institution training and under supervision. The CDC's Biosafety in Microbiological and Biomedical Laboratories (BMBL) explains biosafety levels: https://www.cdc.gov/labs/bmbl/index.html (Checked on 2026-10-23.) Nothing in this course asks you to extract nucleic acids at home.

Common mistakes

• Skipping the dry spin, so ethanol carries over into the eluate.
• Mixing up flow-through and column: discarding the column instead of the flow-through.
• Overloading the column with too much starting material, which lowers purity and yield.
• Touching tube rims and tips during RNA work.
• Trusting A260 alone without checking the ratios.

Quick recap

• Silica methods: lyse, bind (chaotropic salt), wash (ethanol), dry, elute (low salt).
• RNA needs RNase-free technique, speed, cold and usually DNase treatment.
• Check concentration (A260) and purity (A260/A280 about 1.8 DNA or 2.0 RNA; A260/A230 about 2.0 to 2.2).
• Run an extraction blank and record lots and deviations.

Try it

Open the Do tab to annotate a real kit protocol and judge three sets of spectrophotometer readings, on paper.