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Competent Cell Preparation

For n transformations of v volume:

Materials

1. TransformationPrepare enough transformation/storage solution (TSS), ≥nv volume
   TSS: LB 10% (WLysogeny Broth (LB) 10% _m/V)  PEG-3350, 5% (5% _V/V) DMSO   DMSO, 20 mM MgCl₂, pH 6.5):
   For 100 mL TSS : combine, store at 4°C:
   - from non-sterile components:Sterile rich growth medium (+antibiotic if necessary)
   • Dissolve 10 g PEG-3350 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).
     Store at 4°C.
   For 100 mL TSS from sterile components
   • 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. • Store at 4°C.
4. Sterile rich medium
   ≥20nv volume LB
   or ≥7nv volume TB or LB³⁰
5. Culture tubes for seed cultures
6. Culture flasks large enough to hold growth medium volume (detergent-free).
7. Shaking incubator space for the culture flasks.
8. Ice bucket/tray large enough to swirl flasks in. Access to ice.
9. 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.
10. 4°C centrifuge and rotor compatible with centrifuge bottles.
11. A serological pipette at -20°C for adding nv volume TSS.
12. Labeled cryoboxes for comp cell aliquots, prechilled at -80°C.
13. Liquid nitrogen, dewar, and slotted ladle (optional)
14. Consumables 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.
    2. Extra tubes or bag of tubes, for aliquotting any excess comp cells, if desired.
    3. Serological pipettor and few 10 mL pipettes, for resuspending 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. 
    7. Ethanol spray
    8. Paper towels
    9. Extra gloves
    10. Trash bin/bag
        Image Added

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 stock.
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 ≈7nvInoculate 0.8n mL pre-warmed LB with 1% seed culture (or 0.25n mL TB with same). Incubate 37°C , ≈300 rpm.(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.
   Detergent-free glass/plasticware helps efficiency. See note.
   Grow LB culture to OD₆₀₀=0.
3–0
4. 25–0.
4
5. 35 (early exponential phase). For TB
OD₆₀₀=1–2
6. OD₆₀₀≈1–2. It takes between
2–3
7. 1.5–3 hours at 37°C,
300
8. 250 rpm in a large flask.
   • Tenfold less efficiency at OD₆₀₀=0.55.
   • Several tenfold lower-efficiency at stationary phase (10⁵–10⁷/µg DNA), but still fine for transforming pure plasmid or
simple
9. • the simplest cloning.
10. Pre-chill centrifuge bottles that can hold the culture volume, preferably flat-bottom. Clean and pre-chill appropriate rotor and centrifuge for the bottles.
11. Detergent-free glass/plasticware helps efficiency. See note.
12. Label and chill tubes to hold competent cells. Typically 200 µL comp cells are stored per tube, so as to allow 5 transformations/tube.
13. Swirl When in log phase (OD₆₀₀≈0.2–0.35), swirl culture vessel in ice bath for several minutes to rapidly stop growth when OD reached. When culture is ice-cold, decant into the prechilled centrifuge bottles and balance them. Use chilled sterile water if necessary.
14. Centrifuge cells 2500×g, 10 min at 0–4°C.
15. Dump medium and on ice, gently resuspend the cell pellets in Gently decant medium away from pellet, shaking to drain. Return to ice. Add 5% the original LB culture volume of ice-cold TSS (0.04n mL)of 4°C TSS (nv mL) using prechilled serological pipette. For TB culture, 16% volume TSS (0.04n mL)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 into 50 mL conical tubes for easier access with pipette for aliquotting.
16. 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 10-fold concentration in TSS. 20-fold as here, or even 40-fold increases transformation efficiency, but requires larger culture.
17. Aliquot into tubes in the cold room with cold materials (for top efficiency) or on a ice/cold block. You can use a repeater pipette or multichannel pipette from a reservoir if in the cold room.
    Cap , and cut the tubes, and slice apart if using tube strips.
18. Flash freeze in liquid nitrogen, if desired for storage.
    • Ethanol from a dry-ice-ethanol bath fails to dry from tubes/boxes.
19. Quickly move tubes to prechilled, labeled -80°C freezer boxes.

Summary

1. , 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 µL) on ice for a few min.
   Use one per assembly reaction. An aliquot can be split/distributed; 5 µL is sufficient to transform a pure plasmid.
2. Add DNA. Flick five times to mix. Don't vortex or triturate (pipette).
3. Incubate on ice for at least 5 min, but best 30 min.
4. Heat shock 42°, for 30 s, optimally in a water bath.
5. Return to ice immediately for at least 1 min.
6. Recovery: remove from ice and add 160 µL room-temp non-selective recovery medium (e.g. SOC). Not crucial for Amp^R transformation.
   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 for NEB Turbo. Neutral for DH10B and OmniMAX. Somewhat improves transformation efficiency of DB3.1.
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)
6. Thaw cells on ice, or use cold cells straight from comp cell prep.
7. Add 4°C 5× KCM (1:4 KCM:cells). Mix well.
8. Aliquot 50 µL cell-KCM mix into tubes containing DNA. Pipet to mix, or flick/vortex briefly and quickly centrifuge 1 s.
9. Incubate on ice for 5–30 min.
   • Efficiency maximizes by 30 min, 4–5-fold over 5 min incubation.
10. Heat shock at 42°C 45–120 for 30–45 s, optimally in water bath.
    • 30 s optimal for NEB Turbo (3.0E7 CFU/µg in water bath). Efficiencies fell for longer heat shocks: 28% less for 45 s, 31% less for 60 s, and 61% less for 90 s.
    • 45 s purportedly close to optimal for many strains.
    Recover by adding 1–10 volumes rich recovery medium at 37°C, horizontally shaking for 45 min–1.
    • Some strains/preparations/methods might do well with up to 90 s.
    • 78% lower efficiency in metal bead bath than water bath for 45 s heat shock of NEB Turbo.
11. Immediately return to 4°C (ice or cold block) for at least 1 min.
12. Remove from 4°C. Add 1–10 volumes room-temp recovery medium (non-selective).
    • Shyam found that adding 3 volumes (120 µL) SOC to lab aliquot 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 in a 37° horizontal shaker.
    • 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
    5 hr for all antibiotic resistances
    • .
    • SOC and TB/2×YT + glucose/Mg 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.
13. 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.
    • Shyam found that 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.
14. 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.
    • Save resources; use half to a third the number of plates by plating transformations on half/third-sectors of plates. ☮
    • 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.