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Competent Cell Preparation

For n transformations of v volume:

 Materials

1. Transformation/storage solution (TSS), ≥nv volume
   TSS¹: Lysogeny Broth (LB) 10%_m/V PEG-3350, 5%_V/V DMSO, 20 mM MgCl₂, pH 6.5.
   For 50 mL TSS from non-sterile components:
   • Dissolve 5.263 g PEG-3350 in 50 mL LBand
   • Add 1.053 mL 1 M MgCl₂ (0.3103 g MgCl₂·6H₂O )or 1.053 mL 1 M MgCl₂
   • in a final 50 mL LB
   • Adjust pH to 6.5 using NaOH/KOH/HClconcentrated HCl. (pH 6.4–6.8 optimum¹)
   • Filter-sterilize. Transfer 47.5 mL to a sterile tube.
   • Last, add 2.5 mL DMSO (auto-sterile, filter-incompatible).
     Store at 4°C.
   For 50 mL TSS from sterile components (cannot be pH-adjusted to optimum)
   • 42.5 mL LB (autoclaved, RT)
   • 5 g PEG-3350 in 5 mL H₂O H₂O (filter-sterilized)
   • 1 mL 1 M MgCl₂ MgCl₂ (autoclaved, RT)
   • 2.5 mL DMSO (auto-sterile)
     Store at 4°C.
2. Sterile rich growth medium (+antibiotic if necessary)
   ≥20nv volume LB
   or ≥7nv volume TB or LB³⁰
3. Culture tubes for seed cultures
4. Culture flasks large enough to hold growth medium volume (detergent-free).
5. Shaking incubator space for the culture flasks.
6. Ice bucket/tray large enough to swirl flasks in. Access to ice.
7. Chilled centrifuge bottles/lids or tubes appropriate for holding culture volumes and balancing. (detergent-free)
   • Bottles must be able to collectively hold culture(s) without any exceeding ¾ capacity. Filling bottles higher will result in leakage into rotor.
8. Chilled sterile water to balance centrifuge bottles, if needed.
9. 4°C centrifuge and rotor compatible with centrifuge bottles.
10. A serological pipette at -20°C for adding nv volume TSS.
11. Labeled cryoboxes for comp cell aliquots, prechilled at -80°C.
12. (opt.) Liquid nitrogen, dewar, and slotted ladle (optional).
13. Consumables Materials on a mobile lab bench cart for the cold room:
    1. n tubes (e.g. 200 µL tubes) for cell aliquots, labeled/marked and arranged in clean tube racks or tip rack+boxes. Blade, if planning to cut apart tube strips.
    2. Extra tubes or bag of tubes, for aliquotting any excess comp cells, if desired.
    3. Serological pipettor pipette pump/controller and few 10 20 mL pipettes, for resuspending and transferring cells.
    4.  (opt.) Electronic repeater pipette and a 2. 5 mL combitip per strain and some extras. Reservoirs, if opting to us a multichannel pipette.
    5. A 50 mL tube per strain to hold resuspension.
    6. Micropipettor and tips appropriate for aliquot volumes ≥v. . (Sometimes useful to aliquot remaining volume even when using electronic repeater pipette.)
    7. Ethanol spray
    8. Paper towels
    9. Extra gloves
    10. Trash bin/bag
    11. (opt.) If flash freezing, box/bag to collect all tubes in before flash-freezing.
        

Procedure

1. Grow seed culture to saturation: inoculate ≥≈1/100 culture volume of rich medium (+selection, if necessary) with colony or frozen stock chunk, and incubate at growth temp (e.g. 37°C) shaking for ≥≈8 hr for LB.Meanwhile, prepare growth medium and warm to 37°C. 
   • Use a trusted source of the strain. Don't obtain from old plates; retransform necessary plasmid(s) or obtain directly from frozen stock or from plated frozen stockrather fresh plate or healthy agar stab.
2. Meanwhile, prepare growth medium and warm to 37°C.
   Prepare the mobile lab bench cart for the cold room as described, and to cool the materials, incubate the cart at 4°C for at least an hour before cell harvest time (e.g. before next step).
   Set centrifuge to 4°C, allowing an hour for chamber and rotor to cool. (e.g. before next step).
3. Inoculate 20nv mL pre-warmed LB with 0.2% seed culture (or ≈7nv mL TB with same). Incubate 37°C (or optimally 20–33°C), shaking 250 rpm for flasks or 800 rpm for blocks.When in log phase (OD₆₀₀≈0.2–0.35), swirl culture vessel in ice bath for several minutes to rapidly stop growth. When culture is ice-cold, decant into prechilled centrifuge bottles and balance them. Use chilled sterile water if necessary.
   • 37°C works well, used for pre-2018 comp cell batches. But such temperatures (33–37°C) can decrease transformation efficiency 2–10-fold by increasing less-fluid, saturated membrane lipids.
   Grow LB culture to OD₆₀₀=0.2–0.3 35 (early exponential phase). For TB OD₆₀₀≈1–2. It takes between 1.5–3 hours at 37°C, 250 rpm in a large flask, a bit slower at 30°C. Monitor with periodic OD measurement.
   • Transformation efficiency decreases linearly between OD=0.3–0.6.¹
   • Several tenfold lower-efficiency at stationary phase (10⁵–10⁷/µg DNA), but still fine for transforming pure plasmid or the simplest cloning.
4. When in early log phase (OD₆₀₀≈0.2–0.35), swirl culture vessel in ice water bath for several minutes until 5°C to rapidly stop growth. It takes up to 20 min for ~1 L volumes.
5. When culture is ice-cold, decant into prechilled centrifuge bottles and balance them. Use chilled sterile water if necessary.
   Centrifuge cells 2500×1000×g, 10 min at 0–4°C5–20 min (depending on volume) in a prechilled 2–4°C centrifuge.
6. Gently decant medium away from pellet, shaking the bottle to drain. You can use some towels to absorb the last of the medium. Return bottles to drained ice.
   Perform remaining steps with pre-chilled materials in the cold room for maximum efficiency.
7. To the cell pellets, add Add 5% the original culture volume of 4°C TSS (nv mL) using prechilled serological pipette. For TB culture, 14% volume TSS. Keep centrifuge bottles on ice and move to cold room.
   Gently resuspend pellet in TSS using cold serological pipette, or P1000 tip if small volume.
   Decant resuspension For large volumes, decant cells into 50 mL conical tubes on ice for easier access with pipette for aliquotting.
8. Aliquot into tubes in the cold room (for top efficiency) or on a cold block or ice. You can use a repeater pipette or multichannel pipette from a reservoir.
   • Flat-bottom centrifuge bottles allow easier, gentle resuspension of pellets by pipetting against wall.
   • Original protocol instructs 101–10-fold concentration in TSS. 20-fold as here, or even 40-fold increases transformation efficiency, but requires larger culture .
   Aliquot into tubes in the cold room with cold materials (for top efficiency) or on ice/cold block. You can use a repeater pipette or multichannel pipette from a reservoir.
   • with diminishing incremental efficiency.
9. Cap the tubes while they are racked, and slice apart if using tube strips. Minimize touching the tube to keep them cool. Check that all caps are fully in the tube.
   Dislodge tubes from racks into a bag/box without touching the bottom. This is easiest by pushing them out from the bottom using a spare rack or tip box.
10. (opt.) Flash freeze in liquid nitrogen , if desired for storagemaximum efficiency.
    • Ethanol from a dry-ice-ethanol bath fails to dry from tubes/boxes.
11. Quickly move tubes to prechilled, labeled -80°C freezer boxes, either directly from 4°C or directly ladled out of liquid nitrogen dewar. If not freezing, proceed to transformation right away.
    • Flash freezing improves competence, but simply freezing is estimated to reduce competence only a minor fewfold.

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.

ZymoBroth™, containing only 0.5–5% yeast extract and tryptone (LB components) and 10 mM MgCl₂, improves many strains' competence using an unspecified protocol, 13-fold for TG1, the NEB Turbo parent. MgCl₂ in the growth medium may thus be more effective than the 20 mM MgCl₂ in TSS is alone.

Transformation

Summary for frozen lab aliquots

1. Thaw comp cell aliquots (40 50 µL) on ice for a few min.
   Use one per assembly reaction, adding ≤5 µL.
   An aliquot can be split/distributed; 5 µL is sufficient to transform a pure purified plasmid.
2. Add DNA. Flick five times to mix. Don't vortex or triturate (pipette-mix).
3. Incubate on ice for at least 5 ≥5 min, but best 30 min.
4. Heat shock 42° for 30 s in a heat block or water bath.
5. Return to ice immediately for at least 1 1–2 min.
6. Recovery: remove from ice and add 160 150–200 µL room-temp non-selective recovery medium (e.g. SOC). Not crucial for Amp^R transformationFlick to mix.
   Incubate at growth temp (e.g. 37°C) 45–90 min stationary, before plating on warm/RT plates.

Detailed

1. Thaw -80°C comp cell aliquots on ice or cold block for a few min. Or use comp cells within hours of comp cell prep. Keep at 4°C except for heat shock and recovery.
   • Unused volume of comp cells can be frozen back once and reused, albeit with some loss of competence. Lab aliquots are small/abundant enough not to merit this.
2. optional: Add 4°C 5× KCM to 1×.
   
   •  ≈Twofold lower efficiency lower efficiency for NEB Turbo. Neutral for DH10B and OmniMAX. Somewhat improves transformation efficiency of DB3.1 [Shyam].
3. Add DNA, aiming for ≤10% comp cell volume.
4. Flick five times to mix. Do not vortex or triturate.
5. Incubate at 4°C (ice or cold block) for 5–30 min.
   • Efficiency maximizes by 30 min, supposedly.
6. Heat shock at 42°C for 30–45 s in a heated block or water bath.
   • 30 15–30 s optimal for NEB Turbo (3.0E7 CFU/µg in water bath). Efficiencies fell for longer heat shocks: 28% less loss for 45 s, 31% less for 60 s, and 61% less for 90 s [Shyam].
   • 45 s purportedly close to optimal for many strains. Some strains/preparations/methods might do well with up to 90 s.
   • 78% lower loss in efficiency in metal bead bath than water bath for 45 s heat shock of NEB Turbo [Shyam].
   • People on OpenWetware find heat shock after ice incubation improves efficiency 10–20 fold.
     
7. Immediately return to 4°C (ice or cold block) for at least 1 min.
8. Remove from 4°C. Add 1–10 volumes room-temp recovery medium (non-selective). Flick or pipette to mix (trituation may be ok).
   • Adding 3 volumes (120 150 µL) SOC to lab aliquot standard of 40 50 µL NEB Turbo transformations in the original 200 µL tubes, flicking, and incubating stationary 37° incubating stationary 37° gave just as good efficiency as recovering in 10 volumes SOC in a test tube incubated in a 37° horizontal shaker [Shyam].
   • Fewer volumes of recovery medium can prevent having to switch to larger tubes than aliquot tube, but better recovery medium is then probably more important. Efficiency purportedly maximized by approaching 10 volumes recovery medium to improve transformation mix dilution, though shown otherwise.
   • SOC and or TB/2×YT/LB³⁰ + glucose/Mg²⁺ are best; LB+glucose ±Mg²⁺ is good. Media with poor carbon sources (LB, 2×YT) are ok. Mg²⁺, present in part of 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.
9. Incubate at growth temp (often 37°C) for 45 min–1.5 hr, optionally shaking.
   • Lower efficiency obtained with 30 min recovery, no rich medium addition, poor rich medium addition (LB), or sometimes no aeration during recovery.
   • Adding 3 volumes SOC to lab stock standard of 40 µL NEB Turbo transformations in the original 200 µL tubes, flicking, and incubating stationary 37° gave just as good efficiency as recovering in 10 volumes SOC in a test tube incubated 37°C, shaking [Shyam].
   • Transformants of plasmids with temperature-sensitive replicons will typically need to be grown at a lower, permissive temperature.
10. Spread/streak a fraction of the transformation on selective agar medium (plates), optimally room-temp or warmed to growth temperature (often 37°C).
    Store the remainder at 4°C in case later needed due to finding too few or too dense colonies on the transformation plate.
    • Spread or streak only as much as can fit on the plate/sector without either flowing into neighboring sectors or inhibiting streaking dilution such that single colonies are not obtainable (the case with higher efficiency transformations).
    • Save resources; use half to a third the number of plates by plating transformations on half/third-sectors of plates. ☮