The heat/chemical shock transformation method is a quick, economical method for transforming (inducing cell uptake of) self-propagating DNAs (plasmids) and possibly linear non-propagating DNAs under conditions favoring integration into resident DNA.
The CCMB80 method starts by growing cultures at an unusually cool ≈20°C, to minimize membrane stiffness from growth at higher temperatures. Cell culture is cooled to arrest growth in exponential phase, in which the cell wall is least developed, facilitating passage of DNA. The cells are washed and stored in CCMB80 solution, a solution of: the rich nutrient medium LB, glycerol as a cryoprotectant to allow freezing and a viscous agent to reduce DNA diffusion and facilitate binding to the cell, and three divalent cation salts: calcium, manganese, and magnesium chlorides, to neutralize the divalent charge of DNA phosphodiesters and LPS charge and disrupt protein–membrane/LPS interactions that occlude DNA binding. Cold temperature crystallizes the membrane, stabilizing pores called adhesion zones (2). The DNA to be transformed is added and incubated to give time for cation-association and binding and penetrance of cells, facilitated by a heat shock that supposedly creates a temperature differential that induces flow that carries the charge-shielded DNA through adhesion zones. Recovery in non-selective medium allows expression of antibiotic resistance from the transformed DNA, necessary for survival of plated transformants on selective medium.
Transformation efficiency with the CCMB80 method is best for E. coli K-12 str. MC1061/DH10B derivatives, yielding 1×108–109 CFU/µg pUC19 (0.1–1% of plasmid molecules into 1–5% of viable cells).
Based on Tom Knight's OpenWetware adaptation (6) of the Hanahan et al. calcium/manganese-based transformation protocol (5) , which is modified from the Hanahan protocol mainly by omitting the frozen stock preparation on SOB agar and broth, restoring the Mg in SOB, lowering the growth temp to 20°, and doubling the transformation cell concentration. Composed by Shyam Bhakta.
For n transformations of v volume:
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) | Dissolve with |
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, det-free | 10% V/V 20% V/V | 126 g/L 200 mL/L | |
0.1 M HCl, det-free | →pH 6.4 | Adjust pH. | |
Filter-sterilize*. Store at 4°C * Detergent-free membranes (PES, NYL, CN) are preferred for filter-sterilization. |
Allow each salt to dissolve before adding next. 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) .
≥25n v 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 | Dissolve with stir bar. |
Yeast extract | – | 5 g/L | |
NaCl | 10 mM | 0.584 g/L | |
KCl | 2.5 mM | 0.186 g/L | |
Milli-Q water | Reserve Mg soln volumes | ||
NaOH, 1 M | → pH 7.0 | Check pH. typically not needed | |
Autoclave 15 min ≤ 1 L, 20 min 1–1.4 L. Or filter sterilize* after adding components below. | |||
MgCl2 sterile | 10 mM | 10 mL/L 1 M | autoclaved or filtered |
MgSO4 sterile | 10 mM | 10 mL/L 1 M | autoclaved or filtered |
Glucose sterile | 20 mM | 20 mL/L 1 M | For SOC only. Can be spiked-in. |
Store at 4° for best long-term preservation of nutrients. * Detergent-free membranes (PES, NYL, CN) are preferred for filter-sterilization. |
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 H₂ O used to prepare transformation buffers can reduce the efficiency of transformation of competent bacteria. H ₂ O obtained directly from a well-serviced Milli-Q filtration system (Millipore) usually gives good results. If problems should arise, treat the deionized H ₂ O 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."
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) .
We have never used or tried ligation-adjustment buffers, nor have felt the need to, as simply using at most one-tenth the volume of Golden Gate assembly reaction (5 µL) as comp cell volume (50 µL) seems to be reliable enough to get hundreds of colonies.
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.
CaCl2 | 400 mM | 44.392 g/L (·2H2O) |
MnCl2 | 100 mM | 12.584 g/L (·4H2O) |
CH3CO2K (KOAc) | 40 mM | 3.926 g/L |
Glycerol, or 50% Glycerol | 46.8% V / V 20% V / V | 468 mL/L, 126 g/L 200 mL/L (50%) |
0.1 M HOAc | →pH 6.4 | 12.8 mL/L 10% acetic acid |
Filter-sterilize*. Store at 4°C |
A patent from Stratagene(8) describes transformation efficiency being enhanced by adding a final 110 mM NaCl and 50 mM 2-mercaptoethanol (2-sulfanylethanol) 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-sulfanylethanol 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 40%. After comp cells are thawed on ice, add 0.8 µL 2-mercaptoethanol to cells, flick five times to mix, and incubate 10 min on ice before proceeding with transformation and adding DNA.
Hanahan(2) found that 7% DMSO with either 75 mM dithiothreitol or 25 mM 2-sulfanylethanol act as potent transformation enhancers. One might thus surmise that they may have some enhancing effect when added to CCMB80 comp cells upon thawing, perhaps 10–30 min before adding DNA.
Recovery: Incubate at growth temp (often 37°C) for 1 hour, optionally shaking. Recovery time can be reduced at the expense of efficiency.
Meanwhile, you may incubate selective agar plates inverted to warm them.
Thermocycling may somewhat increase transformation efficiency compared to heat shocks performed between cold and heated metal tube blocks. With a simple single heat shock, good competent cells produce hundreds of colonies for even complex assemblies with low yield. However, thermocycling may at times be convenient for more unattended time.
After incubating cells + DNA on ice or cold block, add to thermocycler block prechilled to the first step and proceed to the next step. This way, it's less taxing on the thermocycler, not built for sustained low-temp holds.
Unheated lid | |
1°C | 5–30 min Try to do this cold incubation on ice/cold block instead. |
42°C | 30 s |
1°C | 1–2 min |
42°C | 30 s ; beep |
1°C | 1–5 min ; beep |
END | Add recovery medium. |
Transformation recovery is generally prescribed to consist of adding the transformation suspension to 9–10 volumes prewarmed recovery medium and shaking for 1 hr.
Home-made CCMB comp cells appear to do as well if not better with just 2–3 volumes recovery medium added to the 250 µL "PCR" tubes that transformations are often performed in for space efficiency, thermocycling flexibility, and multichannelability of DNA in tube strips into comp cell aliquots.
50 μL chemical transformation of a Golden Gate assembly, split in half before recovery at 37°, 1 hr.
Left: recovered in 3 volumes SOC in PCR tube, sitting static in bead bath in incubator.
Right: recovered in 9 volumes SOC in 0.6 mL tube, rotating/inverting in incubator.