Overnight cultures & streaking for singles
The two most commonly performed lab tasks. Streak from a glycerol stock or transformation, pick a single, grow overnight, use it for miniprep / induction / archive.
Streaking for single colonies
- Pre-warm a fresh LB+antibiotic plate (5–10 min at 37 °C).
- Sterilise a loop in a flame (or use a fresh sterile inoculating loop).
- Take a small scraping from the source: glycerol stock (without thawing it) OR a single colony from another plate.
- Streak quadrant-by-quadrant:
- Q1: heavy zigzag covering ~1/4 of the plate
- Q2: rotate plate 90°; flame loop, cool, drag through the END of Q1, then zigzag into Q2
- Q3: same as Q2 but starting from Q2
- Q4: same, starting from Q3 — should yield isolated single colonies
- Invert plate. Incubate at 37 °C, 14–18 h. Tape edge if humid.
Overnight liquid culture
- In a 14 mL round-bottom culture tube (or 50 mL conical with vented cap), add 3–5 mL LB.
- Add antibiotic from stock (1 µL of 1000× per 1 mL medium).
- Pick a single colony with a sterile P200 tip. Drop the tip into the tube — no need to remove.
- Cap loosely (allow air exchange) and place in a 37 °C shaking incubator at 220–250 rpm.
- Incubate 14–18 h. By morning the culture should be turbid (OD₆₀₀ > 2).
Volume → flask size
| Culture vol. | Vessel |
|---|---|
| 1–5 mL | 14 mL round-bottom culture tube |
| 10–50 mL | 250 mL Erlenmeyer flask |
| 50–250 mL | 1 L Erlenmeyer (or 2 L for large protein expression) |
| 500 mL+ | 2 L baffled Erlenmeyer or fermenter |
Rule of thumb: culture volume ≤ 1/5 of flask volume for good aeration.
Tips
- Always pick from a single colony — using "swipe of a streak" risks mixed populations.
- Pre-warm flask + medium for cleaner growth in protein-expression cultures.
- Don't grow recA⁺ strains for > 18 h; long stationary phase increases plasmid loss/recombination.
- For low-copy plasmids and BACs, grow up to 24 h.