Chemical/Heat-Shock transformation (CCMB80 method)

Competent Cell Preparation

For n transformations of v volume:

  Materials

1. CCMB80 Buffer, ≥9nv volume 
   10 mM KOAc, 80 mM CaCl₂, 20 mM MnCl₂, 10 mM MgCl₂, 10%_V/V glycerol, pH 6.4.

   CaCl2	80 mM	11.76 g/L (•2H2O)
   MnCl2	20 mM	3.96 g/L (•4H2O)
   MgCl2	10 mM	2.03 g/L (•6H2O)  0.95 g/L (anhyd.)
   CH3CO2K (KOAc)	10 mM	0.98 g/L
   Glycerol, or  50% Glycerol	10% V/V  20% V/V	100 mL/L, 126 g/L  200 mL/L (50%)
   0.1 M HCl	→pH 6.4
   Filter-sterilize*. Store at 4°C

   pH adjustment to 6.4 prevents MnO₂ precipitation. During storage, the pH of the solution drifts down to a final value of 6.1–6.2 but then stabilizes. During prolonged storage, a faint precipitate of a tan-colored substance may appear, but does not seem to affect the efficiency of transformation.⁽⁷⁾

    

2. ≥25nv volume SOB (+antibiotic if necessary)
   20 g/L tryptone, 5 g/L yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄, pH 7.0

   Tryptone		20 g/L
   Yeast Extract		5 g/L
   NaCl	10 mM	0.58 g/L
   KCl	2.5 mM	0.19 g/L
   1 M NaOH	→pH 7.0
   Autoclave. Then add:
   MgCl2 (sterile*)	10 mM	5 mL/L 2 M stock, autoclaved or filtered*
   MgSO4 (sterile*)	10 mM	5 mL/L 2M stock autoclaved or filtered*

   * Detergent-free membranes (PES, NYL, CN) are preferred for filter-sterilization.

    

3. Culture tubes for precultures (detergent-free).
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 18–23°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 80% 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 nv 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 pipettor and few pipettes appropriate for resuspending and transferring cells.
    4. A 5 or 50 mL tube per strain to hold resuspension.
    5.  (opt.) Electronic repeater pipette and a 5 mL combitip per strain and some extras. Reservoirs, if opting to us a multichannel pipette.
    6. Micropipette 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 ≈1/300 culture volume, 0.075nv, SOB (+selection, if necessary) with colonies or scrape of frozen stock, and incubate shaking for –18 hr.
   • Use a fresh, trusted source of the strain. For the most reliable results, inoculate a seed culture from colonies/patch from relatively fresh plates streaked from a frozen stock. Don't obtain from old plates.
   • Incubation of preculture at 37°C seems to be fine for 10⁹/µg efficiency , though 20–23°C is recommended.
   • NEB Turbo grown in 20°C SOB saturates at OD≈3.5 and at 10 in LB³⁰. 
2. Inoculate 25nv mL SOB with ≈0.2% seed culture, to OD₆₀₀≈0.01.
   Incubate 18–22°C, shaking 250 rpm for flasks.
   Grow culture to OD₆₀₀=0.2–0.26 (early exponential phase), 8–12 hr. Periodically monitor OD starting at 7 hr.
   • Transformation efficiency from LB cultures decreases linearly between OD=0.3–0.6.⁽¹⁾ The same might apply here.  Do not allow the culture to grow to an OD₆₀₀ > 0.26 for maximum efficiency.
   • Several tenfold lower-efficiency at stationary phase, but supposedly still fine for transforming pure plasmid or the simplest cloning.
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) ^{[Shyam measured with infrared thermometer]}.
4. When culture reaches OD₆₀₀≈0.2–0.26, immediately harvest cells: decant into prechilled centrifuge bottles and balance them. Use chilled sterile water if necessary.
   Centrifuge 1000×g, 10–15 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. Combine volumes to reduce vessels.
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 Hanahan / CSH 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 gently resuspending any visible remaining cell clumps using P1000 tip if necessary.
   
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 supposedly higher efficiency.
    • Flash freezing improves competence, but simply freezing is not estimated to noticeably reduce competence, maybe within twofold ^[SPB].
    • 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 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.

According to the Cold Spring Harbor protocol ⁽⁷⁾: "Detergents and organic contaminants are strong inhibitors of transformation reactions. To avoid problems caused by residual detergent in glassware, use disposable plastic tubes and flasks wherever possible for preparation and storage of all solutions and media used in transformation. Organic contaminants present in the H₂O used to prepare transformation buffers can reduce the efficiency of transformation of competent bacteria. H₂O obtained directly from a well-serviced Milli-Q filtration system (Millipore) usually gives good results. If problems should arise, treat the deionized H₂O with activated charcoal before use."

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. MgCl₂ in the growth medium may thus be more effective than the 20 mM MgCl₂ 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 Mn²⁺ , Ca²⁺ , 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 Mg²⁺ is not beneficial in the growth medium, and the addition of either DMSO or DTT to the transformation buffer reduces competence ⁽⁵⁾ .

Ligation Adjustment Buffer (5× LAB)

According to the CSH protocol: "Ligation mixtures generally contain high concentrations of DNA and, depending on the termini of the insert and vector, a variety of ligation products: linear and recircularized vector, linear insert, dimers, linear and circular vector:insert recombinants, recirculized vectors, etc. In addition, other components of the ligation mixture (e.g., DNA ligase, polyethylene glycol) may interfere with uptake of DNA by competent bacteria.

"A common mistake is to add too much of the ligation mixture to the competent cells. To avoid problems, use no more than 1 µL of a standard ligation reaction in each transformation assay. Better still,

• precipitate the DNA in the ligation mixture with 2 volumes of ethanol. Recover the DNA by centrifugation and dissolve it in 50 µL of TE (pH 7.6)

• or dilute the ligation mixture fivefold. Use 1 µL of the diluted reaction mixture for transformation

• or adjust the buffer composition of the ligation reaction to more closely resemble that of the CCMB80 buffer by adding 0.2 volumes of ligation adjustment buffer to the ligation mixture immediately before using the products of ligation for transformation. After adjustment, the pH of the ligation mixture should be 6.3–6.5.

40 mM KOAc, 400 mM CaCl₂, 100 mM MnCl₂, 46.8%_V/V glycerol, pH 6.3–6.5 with HOAc.

• Shyam thinks glycerol makes more sense at 30% for 5×, which makes a 10% enzyme volume reaction (5% glycerol) a final 10% glycerol, like CCMB80 buffer.

Potential Transformation Enhancer

A patent from Stratagene⁽⁸⁾ describes transformation efficiency being enhanced by adding a final 110 mM NaCl and 50 mM 2-mercaptoethanol to comp cells and incubating 10 min on ice before adding DNA and further incubating.

Elsewhere, however, NaCl is described as being inhibitory to transformation⁽⁹⁾.

NEB writes⁽¹⁰⁾: Addition of 2-mercaptoethanol to cells at a final 24 mM from a high-purity, sterile 1.5 M stock has been shown to increase the pUC19 transformation efficiency of NEB 5-alpha by 140%. After cells are thawed on ice, add 0.8 µL 2-mercaptoethanol to cells, flick five times to mix, incubate 10 min on ice before proceeding with transformation and adding DNA.

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 30–90 s in a heat block or water bath. (Turbo 30 s, DH10B 45 s)
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.
   • 30 s optimal for NEB Turbo.
   • 45 s optimal for many strains, including DH10B. 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. Move to room-temp rack. Add 1–10 volumes room-temp recovery medium (non-selective). Flick to mix (trituation or gentle vortexing may be ok).
   • Open tubes without touching the insides, which have cells on them. Touching multiple tube lids can cross-contaminate the transformations.
   • Adding 3 volumes (150 µL) SOC to lab standard 50 µL transformation aliquots in the original 250 µL tubes, flicking, and incubating stationary 37° gives about 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 + 10 mM 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 ~tenfold less efficiency without recovery, so don't necessarily require it.

8. Incubate at growth temp (often 37°C) for 45–90 min, optionally shaking. Opt.: Meanwhile, incubate agar plates slightly open to warm and slightly dry them.

   • 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.
   • Prewarming the plate is said to slightly improve transformation efficiency, but the main advantage is the quick absorption of plated cell suspension, which may otherwise flow around the surface when moved. You may want to wait until it is absorbed before inverting the plates. You may leave the plates partially uncovered in the incubator and cover and invert them once dried. However, simply incubating them covered and non-inverted is also an option without any noticeable negative consequence, allowing you to not further monitor the plates.
   • 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. ☮
10. Incubate agar plates inverted in an incubator or room of the appropriate growth temperature until there is sufficient growth.
    • Pickable colonies form with MG1655, JS006, or NEB Turbo in 9–10 hr. Other strains like DH10B, other Rec^– strains, and Mach1 require at least 15–16 hr.

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.