TL;DR: 20X SSC contains 3 M NaCl and 0.3 M sodium citrate at pH 7.0. For 1 L using trisodium citrate dihydrate, dissolve 175.3 g NaCl and 88.2 g trisodium citrate dihydrate, adjust to pH 7.0, then bring to 1 L.
SSC stands for saline-sodium citrate. It is a classic high-ionic-strength buffer used in nucleic-acid hybridisation, Southern/Northern blotting and fluorescence in situ hybridisation (FISH). Hybridisation and wash stringency depend strongly on SSC concentration, temperature and formamide content.
20X SSC composition
- 3 M NaCl
- 0.3 M trisodium citrate
- pH 7.0
20X SSC recipe using trisodium citrate dihydrate
| Final volume | NaCl | Trisodium citrate dihydrate |
|---|---|---|
| 100 mL | 17.53 g | 8.82 g |
| 500 mL | 87.66 g | 44.12 g |
| 1 L | 175.32 g | 88.23 g |
Important: sodium citrate is sold in different hydration states. The masses above are for trisodium citrate dihydrate, MW 294.10. Recalculate if using an anhydrous salt.
Preparation
- Add approximately 700–800 mL purified water to a clean beaker.
- Dissolve the NaCl fully.
- Add trisodium citrate dihydrate and mix until clear.
- Adjust to pH 7.0 with HCl.
- Bring to the final volume with purified water.
- Sterile-filter or autoclave if required by your workflow.
How to dilute 20X SSC
| Working concentration | 20X SSC for 1 L | Water |
|---|---|---|
| 1X | 50 mL | 950 mL |
| 2X | 100 mL | 900 mL |
| 5X | 250 mL | 750 mL |
| 6X | 300 mL | 700 mL |
Why SSC concentration changes hybridisation stringency
Higher salt stabilises nucleic-acid duplexes by screening electrostatic repulsion between negatively charged phosphate backbones. Lowering SSC concentration during washing increases stringency, making mismatched hybrids less stable. Temperature and formamide also change stringency, so SSC cannot be considered in isolation.
SSC in FISH
FISH protocols often use multiple SSC concentrations at different stages: higher ionic strength during hybridisation or initial washing and lower ionic strength for stringent post-hybridisation washes. Use the probe-specific protocol because excessive stringency can remove genuine signal.
Troubleshooting
| Problem | Likely cause | Fix |
|---|---|---|
| Weak FISH signal | Wash too stringent | Increase SSC concentration or reduce wash temperature after validation. |
| High background | Wash not stringent enough | Lower SSC or increase temperature gradually. |
| Incorrect stock concentration | Wrong citrate hydration state | Check reagent formula/MW and recalculate mass. |
Frequently asked questions
What is 1X SSC?
150 mM NaCl and 15 mM sodium citrate.
Why is 6X SSC common in older hybridisation papers?
High-salt SSC stabilises nucleic-acid duplexes, which was useful in membrane-based hybridisation systems. Modern protocols may use different salt/formamide combinations.
Related ABMIUM guides
Primary research references
- Gillespie S, Gillespie D. RNA-DNA hybridization in aqueous and formamide solutions using SSC. Biochem J. 1971.
- Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975.
- Moar MH, Purdom IF, Jones KW. Influence of temperature on the detectability and chromosomal distribution of specific DNA sequences by in situ hybridisation. Chromosoma. 1975.