Based on the Chung et al. protocol1.
Competent Cell Preparation
For n transformations:
- Prepare enough transformation/storage solution (TSS: LB 10% (W/V) PEG-3350, 5% (V/V) DMSO, 20 mM MgCl₂, pH 6.5):
100 mL TSS: combine, store at 4°C:
- 85 mL LB (autoclaved, RT)
- 10 g PEG-3350 in 5 mL H₂O (filter-sterilized)
- 2 mL 1 M MgCl₂ (autoclaved, RT)
- 5 mL DMSO (auto-sterile)
Alternatively:
- dissolve 10 g PEG in 90 mL LB
- add 5 mL 1 M MgCl₂
- adjust pH to 6.5 using NaOH/KOH/HCl
- filter-sterilize
- last, add 5 mL DMSO (auto-sterile, filter-incompatible) - Grow at least 0.01n mL seed culture in LB/TB (+antibiotic if necessary) to saturation.
- Inoculate 0.8n mL pre-warmed LB with 1% seed culture (or 0.25n mL TB with same). Incubate 37°C, ≈300 rpm.
- Detergent-free glass/plasticware helps efficiency. See note.
- Grow LB culture to OD₆₀₀=0.3–0.4 (early exponential phase). For TB OD₆₀₀=1–2. It takes between 2–3 hours at 37°C, 300 rpm.
- Several tenfold lower-efficiency at stationary phase (105–107/µg DNA), but still fine for transforming pure plasmid or simple cloning.
- Pre-chill centrifuge bottles that can hold the culture volume, preferably flat-bottom. Clean and pre-chill appropriate rotor and centrifuge for the bottles.
- Detergent-free glass/plasticware helps efficiency. See note.
- Label and chill tubes to hold competent cells. Typically 200 µL comp cells are stored per tube, so as to allow 5 transformations/tube.
- Swirl culture vessel in ice bath to stop growth when OD reached. When culture is ice-cold, decant into the bottles and balance them. Use sterile water if necessary.
- Centrifuge cells 2500×g, 10 min.
- Dump medium and on ice, gently resuspend the cell pellets in 5% the original LB culture volume of ice-cold TSS (0.04n mL). For TB culture, 16% volume TSS (0.04n mL).
- Flat-bottom centrifuge bottles allow easier, gentle resuspension of pellets by pipetting against wall.
- Aliquot into tubes in the cold room (for top efficiency) or on a cold block. You can multichannel pipette from a reservoir if in the cold room.
Cap, and cut the tubes, if using tube strips. - Flash freeze if desired. Quickly move tubes to -80°C freezer boxes.
Summary
Grow at least 0.01n seed culture. Inoculate 1% into 0.8n mL LB or 0.25 n mL TB culture. Resuspend early-log phase LB culture in 0.04n mL cold TSS; TB in 0.12n TSS. Or stationary phase for lower efficiency. Freeze.
Detergent Residue
According to Tom Knight: Detergent is a major inhibitor of competent cell growth and transformation. Glass and plastic must be detergent-free for these protocols. The easiest way to do this is to avoid washing glassware and simply rinse it out. Autoclaving glassware filled ¾ with deionized water is an effective way to remove most detergent residue. Media and buffers should be prepared in detergent-free glassware and cultures should be grown in detergent-free glassware.
Choice of Culture Medium
Shyam found that competent cells prepared from a TB culture produced far more satellite colonies in a transformation of pUC19, compared to competent cells prepared from an LB culture. The effect was mostly apparent after plates sat at room temperature for several days.
Transformation
- Thaw cells on ice, or use cold cells straight from comp cell prep.
- Add 4°C 5× KCM (1:4 KCM:cells). Mix well.
- Aliquot 50 µL cell-KCM mix into tubes containing DNA. Pipet to mix, or flick/vortex briefly and quickly centrifuge 1 s.
- Incubate on ice for 5–30 min.
- Efficiency maximizes by 30 min, 4–5-fold over 5 min incubation.
- Heat shock at 42°C 45–120 s.
- 45 s optimal for many strains.
- Recover by adding 1–10 volumes rich recovery medium at 37°C, horizontally shaking for 45 min–1.5 hr for all antibiotic resistances.
- SOC and TB/2×YT + glucose are best; LB+glucose is a fewfold worse, but good. Media with poor carbon sources (LB, 2×YT) are ok. Mg²⁺, present in SOC, is said to improve efficiency by stabilizing outer membrane.
- β-lactam resistance transformations (ampicillin, carbenicillin) have good efficiency without recovery, but still benefit from it.
- Lower efficiency obtained with 30 min recovery, no rich medium addition, poor rich medium addition (LB), or no aeration during recovery.
- 1–2 volumes recovery medium can save you from having to switch to larger tubes if using thermocycler tubes, but richer medium and aeration probably become more important.
- Efficiency maximized by approaching 10 volumes recovery medium in 1.5–2 mL tube to improve transformation mix dilution and aeration when horizontally shaken.
Original Protocol from Literature
From Chung et al. 1
- Prepare transformation/storage solution (TSS), if not already prepared. (LB, 10% (W/V) PEG-3350, 5% (V/V) DMSO, 20 mM MgCl₂, pH 6.5)
100 mL TSS:
- 85 mL LB (autoclaved, RT)
- 10 g PEG (polyethylene glycol)-3350 in 5 mL H₂O (filter-sterilized)
- 2 mL of 1 M MgCl₂ (autoclaved, RT)
Filter sterilize. Then add…
- 5 mL DMSO (auto-sterile). Store at 4°C. - Grow seed culture.
- Inoculate prewarmed LB 1:100 with saturated culture, and incubate 37°C, 225 rpm to an OD600 0.3–0.4.
- Tenfold less efficiency at OD600 0.55.
- Several tenfold lower efficiency in stationary phase, but still ok.
- Concentrate culture in a cold centrifuge bottle/centrifuge to 1∕10 volume in ice-cold TSS.
- For long-term storage, freeze cells immediately in a dry ice/ethanol bath, and store at -80°C.
- For transformation, pipet 100 µL into a cold polypropylene tube containing 1 µL (100 pg) plasmid. Mix gently. (When frozen cells are used, cells are thawed slowly on ice and used immediately.)
- Incubate cell/DNA mixture 5–60 min at 4°C.
- Efficiency maximizes by 30 min, 4–5-fold over 5 min incubation.
- People on OpenWetware find heat shock after ice incubation improves efficiency 10–20 fold.
- Allow cell recovery in equal volume TSS (or LB 20 mM glucose).