Heat/chemical shock transformation (TSS method)

Competent Cell Preparation

For n transformations:

1. 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)
2. Grow at least 0.01n mL seed culture in LB/TB (+antibiotic if necessary) to saturation.
3. 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.
4. 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.
5. Label and chill tubes to hold competent cells. Typically 200 µL comp cells are stored per tube, so as to allow 5 transformations/tube.
6. 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.
7. Centrifuge cells 2500×g, 10 min.
8. 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.
9. 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.
10. 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

1. Thaw cells on ice, or use cold cells straight from comp cell prep.
2. Add 4°C 5× KCM (1:4 KCM:cells). Mix well.
3. Aliquot 50 µL cell-KCM mix into tubes containing DNA. Pipet to mix, or flick/vortex briefly and quickly centrifuge 1 s.
4. Incubate on ice for 5–30 min.
   • Efficiency maximizes by 30 min, 4–5-fold over 5 min incubation.
5. Heat shock at 42°C 45–120 s.
   
   • 45 s optimal for many strains.
6. 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.