Chemical/Heat-Shock transformation (CCMB80 method)

Competent Cell Preparation

For n transformations of v volume:

  Materials

1. CCMB80 Buffer, ≥n v volume 
   10 mM KOAc, 80 mM CaCl₂, 20 mM MnCl₂, 10 mM MgCl₂, 10% glycerol, pH 6.4.

   CaCl2	80 mM	11.76 g/L (•2H2O)
   MnCl2	20 mM	3.96 g/L (•4H 2 O)
   MgCl2	10 mM	2.03 g/L (•6H 2 O) 0.95 g/L (anhyd.)
   KOAc	10 mM	0.98 g/L
   50% Glycerol 100% Glycerol	20% V/V 10% V/V	50%: 226 g/L 100%: 126 g/L
   0.1 M HCl	→pH 6.4
   Filter-sterilize. Store at 4°C

2. ≥25n v volume SOB (+antibiotic if necessary)
3. Culture tubes for precultures
4. Culture flasks large enough to hold growth medium volume (detergent-free).
5. Refrigerated shaking incubator space for the culture flasks.
   • Only a refrigerated shaker can sustain 20–23°C temperature.
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 multiple centrifuge bottles, if needed.
9. 4°C centrifuge and rotor compatible with centrifuge bottles.
10. (opt.) A serological pipette at -20°C for adding n v volume CCMB80 before going to cold room.
11. Labeled cryoboxes for comp cell aliquots, prechilled at -80°C.
12. (opt.) Liquid nitrogen, dewar, and slotted ladle, if flash freezing.
13. 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 pipette pump/controller and few 20 mL pipettes, for resuspending and transferring cells.
    4.  (opt.) Electronic repeater pipette and a 5 mL combitip per strain and some extras. Reservoirs, if opting to us a multichannel pipette.
    5. A 5 or 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 preculture to saturation: inoculate ≥≈1/100 culture volume of rich medium (+selection, if necessary) with colony or frozen stock chunk, and incubate shaking for ≥≈8 hr for LB.  Meanwhile, prepare growth medium. 
   • Use a fresh, trusted source of the strain. For the most reliable results, inoculate a seed culture from colonies from relatively fresh plates streaked from a frozen stock. Don't obtain from old plates; retransform necessary plasmid(s) or obtain directly from a frozen stock.
   • Incubation of preculture at 37°C seems to be fine for 10⁹/µg efficiency , though 20–23°C is recommended.
2. Inoculate 25nv mL SOB with ≈0.2% seed culture.
   Incubate 20–23°C, shaking 250 rpm for flasks or 800 rpm for blocks.
   Grow culture to OD₆₀₀=0.2–0.35 (early exponential phase), 12–16 hr. Monitor with periodic OD measurement.
   • Transformation efficiency from LB cultures decreases linearly between OD=0.3–0.6.⁽¹⁾ Same might apply here. 
   • Several tenfold lower-efficiency at stationary phase, but supposedly still fine for transforming pure plasmid or the simplest cloning, supposedly.
3. Prepare the mobile lab bench cart for the cold room as described. Prechill both the mobile lab bench cart (with its contents) and the centrifuge to 4°C ≥45 min before cell harvest (next step).
4. When culture is in early log-phase (OD₆₀₀≈0.2–0.35), immediately harvest cells: decant into prechilled centrifuge bottles and balance them. Use chilled sterile water if necessary.
   Centrifuge 1000×g, 10–20 min (depending on volume) in a prechilled 4°C centrifuge.
5. Check for a small pellet. Gently decant medium away from pellet, shaking the bottle to drain. Absorb the last of the medium on the lip with a paper towel. Return bottles to ice.
   
6. Gently resuspend cells in ⅓ culture volume (8nv mL) of chilled CCMB80 buffer, by swirling and striking.
7. Incubate suspension on ice 20 min.
8. Harvest cells as before (step 4–5).
9. Gently resuspend in 1/25 volume (nv mL) chilled CCMB80 buffer by swirling and striking.
   
   • Flat-bottom centrifuge bottles allow easier gentle resuspension of pellets.
   • Original protocol instructs 12-fold concentration in CCMB80 buffer. The OpenWetware version instructs 25-fold concentration.
   
   Perform remaining steps with pre-chilled materials in the cold room for maximum efficiency.
   Decant or pipette cells into 5 or 50 mL conical tubes on ice for easier access with pipette for aliquotting. Finish resuspending any visible remaining cell clumps using P1000 tip.
   
10. Aliquot into tubes in the cold room (for top efficiency) or on cold block or ice. You can use a repeater pipette or multichannel pipette from a chilled reservoir.
11. Cap the tubes while they are racked, and slice apart if using tube strips. Minimize touching the tubes 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 (your hands are warm). This is easiest by pushing them out from the bottom using a spare rack or tip box.
12. (opt.) Flash freeze in liquid nitrogen for maximum efficiency.
    • Flash freezing improves competence, but simply freezing is not estimated to noticeably reduce competence, maybe within twofold.
    • Residual ethanol from a dry-ice-ethanol bath fails to dry from tubes/boxes at -80°C. Freeze tubes in a bag if using this method, and pour tubes into box.
13. Quickly move tubes to prechilled, labeled -80°C freezer boxes, either directly from 4°C, or if flash freezing, directly ladled out of liquid nitrogen dewar or dry-ice/ethanol bag (so as not to heat tubes).
    If not freezing, proceed to transformation right away.

Detergent Residue

According to  Tom Knight⁽⁴⁾: 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 nearly filled 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

ZymoBroth™ ⁽³⁾ , 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. MgCl ₂ in the growth medium may thus be more effective than the 20 mM MgC ₂ in TSS is alone. Even before, Hanahan (1983)⁽²⁾  found that "the presence of 10 to 20 mM Mg²⁺ in all growth media considerably stimulates transformation efficiency," as well as "incubation of the cells at 0°C in a solution of Mn ²⁺ , Ca ²⁺ , Rb ⁺  or K ⁺ , dimethyl sulfoxide, dithiothreitol, and hexamine cobalt(III)."

These enhancements were later found to be more specific to strain MC1061 and derivatives like DH10B, and not strains derived from  Hoffman Berling strain 11008 ( e.g. , MM294, DH1, DH5) as well as from many other strains ( e.g. , HB101, C600), for which Mg ²⁺  is  not  beneficial in the growth medium, and the addition of either DMSO or DTT to the transformation buffer reduces competence⁽⁵⁾.

Transformation

Summary for frozen lab aliquots

1. Thaw comp cell aliquots (50 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 purified plasmid.
2. Add DNA ≤5 µL. Flick five times to mix. Don't vortex or triturate (pipette-mix).
3. Incubate on ice for ≥5 min, best 30 min.
4. Heat shock 42° for 45–90 s in a heat block or water bath.
5. Return to ice immediately for 2 min.
6. Recovery: remove from ice and add 150–200 µL SOC. Flick to mix.
   Incubate at growth temp (e.g. 37°C) 45–90 min stationary.
7. Plate.

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. Add DNA, aiming for ≤10% comp cell volume.
3. Flick five times to mix. Do not vortex or triturate.
4. Incubate at 4°C (ice or cold block) for 5–30 min.
   • Efficiency maximizes by 30 min, supposedly.
5. Heat shock at 42°C for 45–90 s in a heated block or water bath.
   • 45 s supposedly close to optimal for many strains. Some strains/preparations/methods might do well with up to 90 s.
6. Immediately return to 4°C (ice or cold block) for 2 min.
7. Remove from 4°C. Add 1–10 volumes room-temp recovery medium (non-selective). Flick to mix (trituation or gentle vortexing may be ok).
   • Adding 3 volumes (150 µL) SOC to lab aliquot standard of 50 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 [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 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⁺, 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.

8. Incubate at growth temp (often 37°C) for 45–90 min, optionally shaking.

   • Lower efficiency obtained with 30 min recovery, no rich medium addition, poor rich medium addition (LB), or sometimes no aeration during recovery.
   • Transformants of plasmids with temperature-sensitive replicons will typically need to be grown at a lower, permissive temperature.
9. 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. ☮

Colony Transformation

Not recommended for transforming DNA assemblies, but useful for transforming purified plasmids.

1. Pick several colonies or a clump of cells using a pipette tip, toothpick, or inoculation loop.
2. Disperse cells into thawed aliquot of chilled CCMB80, TFB, or FSB.
3. Incubate the cells on ice for 10 min.
4. Add DNA solution (10–1000 ng) in less than 10% volume. Flick to mix.
5. Incubate the cell/DNA solution on ice for 10 min.
6. Heat-shock 37–42°C for 90 s. (Optional if >100 ng DNA used.)
7. Add 2–4 volumes SOC medium and incubate 20–60 min at 37°C. (Optional if >100 ng DNA used.)
8. Plate on selective agar, and incubate to establish colonies.