A silica spin column is the small plastic column, with a silica membrane at its base, that sits inside a 2 mL collection tube in most DNA and RNA extraction kits. Nucleic acid sticks to the membrane under one set of buffer conditions, contaminants are washed through, and clean DNA or RNA is released under another. This page explains the chemistry behind each step, so you can tell why a protocol works and what to change when yield or purity drops.
Key points
- Silica binds DNA and RNA in the presence of a high concentration of chaotropic salt, such as guanidinium thiocyanate or guanidine hydrochloride (Boom et al., 1990).
- The same chaotropic salt lyses cells and inactivates nucleases, which is why lysis and binding happen in one buffer.
- Ethanol or isopropanol added to the lysate strengthens binding and lets more nucleic acid, especially RNA and short fragments, adsorb.
- Binding works best at neutral to slightly acidic pH; ethanol-based wash buffers remove salts and proteins while the nucleic acid stays on the membrane.
- Elution uses a low-salt, slightly alkaline solution (water, 10 mM Tris or TE); removing the salt reverses the conditions that held the nucleic acid on the silica.
- Leftover ethanol or guanidine salt in the eluate is the most common cause of a low A260/A230 and of inhibited RT-qPCR.
How does silica bind nucleic acids?
Under normal conditions both silica (negatively charged silanol groups) and DNA/RNA (negatively charged phosphate backbone) are surrounded by tightly bound water, so they repel each other. A high concentration of chaotropic salt disrupts that hydration shell, and cations in the buffer shield the negative charges. With the water layer and charge repulsion reduced, the phosphate backbone can adsorb directly onto the silica surface. Boom and colleagues described this guanidinium-thiocyanate-plus-silica approach in 1990 and showed it purified double-stranded DNA, single-stranded DNA and rRNA from clinical specimens in under an hour.
Why add ethanol before loading the column?
Ethanol lowers the water activity of the lysate further, so binding becomes stronger and less size-dependent. That is why RNA kits ask you to add ethanol to the homogenate before loading, and why changing the ethanol volume changes which RNA sizes are kept: more ethanol retains more small RNA, less ethanol favours larger RNA.
What does pH do?
Binding is most efficient at neutral to slightly acidic pH, where the silica surface carries fewer negative charges. Elution is most efficient at neutral to slightly alkaline pH, where silica and nucleic acid repel again. If yield is low, check that the lysate was not made alkaline and that the elution buffer is not acidic: unbuffered water can be slightly acidic and elutes less efficiently than 10 mM Tris pH 8–8.5.
What happens in the wash steps?
Wash buffers contain ethanol, so the nucleic acid stays dehydrated and bound while proteins, polysaccharides and most of the guanidine salt are washed through. A typical protocol uses one wash that still contains some chaotropic salt (to strip protein) and one ethanol wash without it (to remove the salt). An extra “dry spin” after the last wash removes residual ethanol, which otherwise carries into the eluate and inhibits downstream enzymes.
Why does low-salt elution release the DNA or RNA?
When water or a low-salt buffer reaches the membrane, the hydration shell returns and the charge shielding disappears, so the nucleic acid leaves the silica. Let the elution buffer sit on the membrane for 1–2 minutes before spinning, and apply it to the centre of the membrane rather than the wall. For DNA, pre-warming the elution buffer can increase recovery of large fragments; keep RNA elutions in RNase-free water.
Troubleshooting by symptom
| Symptom | Likely cause in the column chemistry | What to change |
|---|---|---|
| Low yield | Too little chaotropic salt or ethanol at binding; acidic elution water; membrane overloaded | Follow lysate:ethanol ratios exactly; elute with 10 mM Tris pH 8–8.5; reduce input |
| Low A260/A230 | Guanidine salt carryover | Do both washes; avoid touching the column to the flow-through; add a dry spin |
| RT-qPCR inhibited | Residual ethanol or salt | Dry spin 1–2 min; transfer column without splashing |
| Genomic DNA in RNA prep | DNA co-binds under the same conditions | Use an on-column DNase step or a gDNA-removal column |
| Small RNA lost | Not enough ethanol at binding | Use a kit or protocol designed to retain small RNA |
After elution, check purity with absorbance ratios and integrity on a gel or electropherogram: see RNA quality reference values and how to read A260/A280.
Spin column vs magnetic beads vs phenol–chloroform
- Silica spin column: fast, no organic solvents, easy to run a few to a few dozen samples by hand.
- Silica magnetic beads: the same chemistry on a bead surface; suited to automation and 96-well formats.
- Phenol–chloroform (TRIzol-type): handles difficult or very high-input samples, but uses hazardous organics and is more prone to phenol carryover. Some kits combine both, for example TRIzol-style lysis followed by a silica column.
Changyu Bio silica spin column kits
Changyu Bio supplies silica-membrane spin column kits for total RNA (e.g. CYRN28 Plus RNA Mini Kit), plant RNA, viral RNA, PCR clean-up (CYDR02) and gel extraction (CYDR01). Browse all RNA/DNA extraction kits or request a quote. Research Use Only.
Sources
- Boom R, Sol CJ, Salimans MM, Jansen CL, Wertheim-van Dillen PM, van der Noordaa J. Rapid and simple method for purification of nucleic acids. J Clin Microbiol. 1990;28(3):495–503. doi:10.1128/jcm.28.3.495-503.1990.
- Vogelstein B, Gillespie D. Preparative and analytical purification of DNA from agarose. Proc Natl Acad Sci USA. 1979;76(2):615–619.
Need empty columns for your own buffers? See silica membrane spin columns (Mini & Micro).