- Document
- CT-03
- Section
- Containers, labels & contamination
- Revised
- Sources checked
- Bands
- −80 °C−20 °C
Aliquoting: Split Once, Thaw Once
Dividing stock into single-use portions is the most effective way to protect peptide solutions. How to size aliquots, which tubes to use, and how to keep track of them.
Bench card
- Each aliquot should hold what one experiment uses, so none is thawed twice.
- Do all the dispensing in one session from one freshly prepared solution.
- Pre-label tubes before you start dispensing.
- Freeze promptly, and use −80 °C for long holds or sensitive peptides.
- Record how many aliquots exist and where; remove each one from the inventory when used.
Aliquoting means dividing a stock into portions so that each can be used once. It protects against repeated freezing and thawing, limits contamination from repeated withdrawals, and means an accident affects one tube rather than the whole stock.
When to aliquot
- Any peptide solution that will be stored frozen.
- Lyophilized material that would otherwise be opened many times.
- Expensive or scarce material where a single mistake would be costly.
Sizing aliquots
Work backwards from the experiment. If each assay uses 20 µL of a 1 mg/mL stock, aliquots of 25–30 µL leave a little margin without much waste. Very small volumes, below about 10 µL, are hard to pipette accurately and have a high surface-to-volume ratio, so peptide loss to the tube wall becomes significant. If that happens, use a more concentrated stock or slightly larger aliquots.
| Use per experiment | Suggested aliquot | Tube |
|---|---|---|
| Under 10 µL | 15–25 µL | 0.5 mL low-binding |
| 10–50 µL | Use + 20% | 0.5 mL low-binding |
| 50–500 µL | Use + 10% | 1.5 mL or 2 mL screw-cap |
CL-03
Aliquoting a stock
Split once, at the start, so no portion is thawed or opened twice.
Step by step
- Prepare the solution following Research Solution Preparation.
- Label all tubes first with compound, lot, aliquot ID, concentration and date.
- Arrange tubes on a cold rack in the order of their IDs.
- Dispense steadily with a fresh tip if the solution must remain sterile, otherwise with a single tip, closing each tube as you go.
- Freeze promptly in a labeled box in the target band.
- Update the record: number of aliquots, volume, box and positions.
Sterility
For cell work, prepare and dispense in a biosafety cabinet using sterile tubes and tips, and sterile-filter the stock with a low-protein-binding 0.2 µm filter if needed. Aseptic technique principles, such as minimizing open time and avoiding passing hands over open tubes, apply even in a research setting.
Using an aliquot
Take out one tube, thaw it on ice or at room temperature, mix gently, spin down briefly, and use it. Discard any remainder. Do not return a thawed aliquot to the freezer, and cross it off the inventory.
References
- Cao E, Chen Y, Cui Z, Foster PR. Effect of freezing and thawing rates on denaturation of proteins in aqueous solutions. Biotechnol Bioeng. 2003;82(6):684–690. DOI
- Bhatnagar BS, Bogner RH, Pikal MJ. Protein stability during freezing: separation of stresses and mechanisms of protein stabilization. Pharm Dev Technol. 2007;12(5):505–523. DOI
- Kristensen K, Henriksen JR, Andresen TL. Adsorption of cationic peptides to solid surfaces of glass and plastic. PLoS One. 2015;10(5):e0122419. DOI
- US FDA. Sterile Drug Products Produced by Aseptic Processing — Current Good Manufacturing Practice. Guidance for Industry; 2004. FDA