Competent Cells Support - Getting Started

Table of cap color/strain

ME = max efficiency, SE = subcloning efficiency, LE = library efficiency

Our Mach1™-T1R competent cells grow faster than any of our common cloning strains. It has a doubling time of 54 minutes versus doubling times in excess of 70 mins for standard cloning strains, such as DH5α™ cells. Colonies of Mach1™-T1R begin to be visible on a plate 8 hours after plating the transformation mix at 37 degrees C. It can be mini-prepped from 1.5 mL cultures in as little as 4 hours at 37 degrees C after inoculation with a single large overnight colony.

OmniMAX™ 2 is the preferred strain for transforming libraries because of its high transformation efficiency and genomic cloning compatibility characteristics.

Yes, our INV110 strain is dcm/dam– .

The table below compares the Stbl2™ and Stbl4™ competent cell strains.

Please use our ccdB Survival™ 2 T1R competent cell strain (Cat. No. A10460).

Please see the links below for competent cell recommendations:

• Routine cloning
• Protein expression
• Library construction
• High-throughput cloning
• Cloning unstable DNA

We would recommend a mcr/mrr– strain, which prevents restriction of methylated eukaryotic DNA in the E. coli host. We would also recommend using a T1R strain, as T1 is a common contaminant in genomic/cDNA libraries.

DH5α™ cells are commonly used for routine cloning, but are mcr/mrr+, and therefore not recommended for genomic cloning. The TOP10 competent cells, on the other hand, contain mutated mcr/mrr, and therefore are a good choice for routine cloning and can be used for cloning of methylated DNA, such as eukaryotic genomic DNA. Our Mach1™ strain is the fastest growing cloning strain that is T1 phage resistant.

We would recommend our Stbl3™ competent cells, as they have been tested for cloning of unstable lentiviral DNA sequences containing direct repeats.

There are a few exceptions, but in general the difference is in guaranteed transformation efficiency as follows:

Subcloning Efficiency™ cells are guaranteed to produce at least 1.0 x 10E6 transformants per µg of transformed pUC19 or pUC18 supercoiled plasmid.

Library Efficiency™ cells are guaranteed to produce at least 1.0 x 10E8 transformants per µg pUC19 or pUC18 DNA.

MAX Efficiency™ cells are guaranteed to produce at least 1.0 x 10E9 transformants per µg pUC19 or pUC18 DNA.

Efficiencies of Invitrogen E. Coli host strains

We would recommend the use of our ElectroMAX™ DH12S™ cells (Cat. No. 18312-017). These are electrocompetent cells that are endA+, allowing for production of ssDNA.

Yes, we would recommend using our MAX Efficiency® DH10Bac™ competent cells. The DH10Bac™ E. coli strain contains a bacmid, which can recombine with our donor plasmid, pFastBac, to create an expression bacmid. These cells are a part of our Bac-to-Bac® Baculovirus Expression System.

The only difference between TOP10 and TOP10F’ cells is that the latter contain the F’ episome that carries the tetracycline resistance gene and allows isolation of single-stranded DNA from vectors that have an f1 origin of replication. The F’ episome also carries the lacIq repressor for inducible expression from trc, tac, and lac promoters using IPTG. TOP10F’ cells require IPTG induction for blue/white screening.

Strains that contain an F plasmid, such as TOP10F’, are not recommended for transformation and selection of recombinant clones in any TOPO® vector containing the ccdB gene. While the F plasmid does encode the CcdA protein, which acts as an inhibitor of the CcdB gyrase-toxin protein, the half-life of the CcdA protein is shorter than that of the CcdB protein. Overexpression of the CcdB protein causes cell death when its action is not prevented by sufficient CcdA.

Competent cell efficiency is measured by transformation efficiency. Transformation efficiency is equal to the number of transformants, or colony forming units, per µg of plasmid DNA (cfu/ µg).

In plates, we recommend 50 µg/mL X-gal and 1 mM IPTG (0.24 mg/mL). When spreading directly onto agar plates, we recommend 40–50 µl of 40 mg/mL X-gal (2% stock) in dimethylformamide and 30–40 µl of 100 mM IPTG on top of the agar. Let the X-gal and IPTG diffuse into the agar for approximately 1 hour. Do not plate on media containing glucose, as it competes with X-gal or bluo-gal and prevents cells from turning blue.

Yes, this is possible. We recommend using saturating amounts of DNA (10 ng of each plasmid). Make sure that the origin of replication is different in each plasmid so that they can both be maintained in the cell. If the ori is the same, the plasmids will compete for replication and the one with even a slight disadvantage will be lost. Alternatively, cells with a resident plasmid can be electroporated with a second plasmid without “electrocuring” taking place.

For long-term storage, preparation of glycerol stocks stored at –70 degrees C is recommended. Follow the protocol below:

1. Pick one colony into 5 mL LB broth or S.O.C. medium. Grow overnight at 37 degrees C.
2. Prepare glycerol solution: 6 mL of S.O.B. medium and 4 mL of glycerol.
3. Take one volume of cells and add one volume of glycerol solution and mix.
4. Freeze in ethanol/dry ice. Store at –70 degrees C.

S.O.B. Medium (per liter)

We recommend using a device that applies 16 kV/cm and the appropriate conditions listed in the manual for each electrocompetent strain.

Please see the list below of restriction enzymes sensitive to dam or dcm methylation:

• Dam: Bcl I, Cla I, Hph I, Mbo I, Mbo II, Taq I, Xba I, BspH I, Nde II, Nru I
• Dcm: Ava II, EcoO 109 I, EcoR II, Sau96 I, ScrF, Stu I, Aat I, Apa I, Bal I, Kpn I, ISfi I

1. Use pUC or pUC-based vectors that contain the portion of the lacZ gene that allows for α complementation.
2. Select an E. coli strain that carries the lacZdeltaM15 marker.
3. Plate transformations on plates containing X-gal. Spread 50 µg of 2% X-gal or 100 microliters of 2% bluo-gal (both can be dissolved in DMF or 50:50 mixture of DMSO:water) on the surface of a 100 mm plate and let dry. Alternatively, add directly to the cooled medium (~50 degrees C) before pouring the plates at a final concentration of 50 µg/mL for X-gal and 300 µg/mL for bluo-gal. Plates are stable for 4 weeks at 4 degrees C.
4. If the strain used carries the lacIq marker, add IPTG to induce the lac promoter. Spread 30 µl of 100 mM IPTG (in water) on 100 mm plates. Alternatively, add the IPTG directly to cooled medium (~50 degrees C) before pouring the plates to a final concentration of 1 mM. Plates are stable for 4 weeks at 4 degrees C.
5. Do not plate E. coli on medium containing glucose if using X-gal or bluo-gal for blue-white screening. Glucose competes as a substrate and prevents cells from turning blue.

Please view our bacterial growth media chart.

To achieve the highest possible transformation efficiency with Thermo Scientific DH5α (Cat. No. EC0112) and DH10B (Cat. No. EC0113) competent cells, please follow the transformation protocol provided with the product and pay attention to the following details:

- Make sure to use wet well-pressed ice to maximize cold surface volume in all the transformation steps.
- Perform the steps quickly and accurately.
- Use chilled microcentrifuge tubes and bury the whole tube up to the cap in the wet ice.
- Do not exceed the recommended heat shock time and transfer the tube with cells back to the wet ice immediately.

Preparation procedures and formulations for all of our competent cells are proprietary. All chemically competent cells are delivered in an aqueous solution that contains a mixture of salts, along with a freezing stabilizer such as glycerol or DMSO.

Please see the Certificate of Analysis for your specific product. In general, all competent cells are tested for transformation efficiency with pUC19 supercoiled plasmid under nonsaturating conditions (10–500 pg per reaction depending on product). Cells are also screened for resistance to ampicillin, kanamycin, tetracycline, chloramphenicol, streptomycin, zeocin and spectinomycin, and for absence of bacteriophage contamination. Some products may undergo additional screening depending on the applications they are used for. Tests may include transformation efficiency with another vector specific to the application (such as pDONR transformations with ccdB Survival™ 2T1R resistant cells), screening for auxotrophic markers, metabolic markers, phage resistance, etc.

We do not recommend storing competent E. coli strains in liquid nitrogen as the extreme temperature can be harmful to the cells. Also, the plastic storage vials are not intended to withstand the extreme temperature and may crack or break.

Extremes in pH and/or high ionic strength will inhibit the activity of Zeocin™ antibiotic. To optimize Zeocin™ antibiotic selection in E. coli, the salt concentration in the growth medium must be < 110 mM and the pH must be 7.5. In particular, a low salt LB formulation should be used for Zeocin™ antibiotic selection (containing 5 g or less of NaCl per Liter).

Also, any E. coli strain that contains the complete Tn5 transposable element (e.g., DH5αF'IQ™, SURE, SURE2) encodes the ble (bleomycin) resistance gene. These strains can confer resistance to Zeocin™ antibiotic. For the most efficient Zeocin™ antibiotic selection, use an E. coli strain that does not contain the Tn5 gene (e.g., TOP10, DH5a™, DH10B™, etc.).

One Shot® format is supplied as pre-aliquoted tubes, each tube sufficient for a single reaction. Using One Shot® cells you can avoid freeze-thaws and extra pipetting steps. Standard format is what is supplied in general use kits designed for scientists who perform more than one transformation at a time—each vial contains sufficient volume for two or more reactions. MultiShot™ kits are for high-throughput transformations, and are supplied in 96-well thin-wall polycarbonate microtiter plates.

The MultiShot StripWell format is supplied as pre-aliquoted competent cells in 96 tubes contained in a rack. The tubes are attached in strips of 8 tubes, and there are 12 strips that contain 50 µL of chemically competent cells. This format offers the most flexibility to transform 1 tube or up to all 96 tubes at once and is intended for varied or medium-throughput use.

The MultiShot FlexPlate format is supplied as pre-aliquoted competent cells in a 96-well PCR plate. Each well contains 20 µL of competent cells. The plate can be separated at each column of 8 wells, offering a high level of flexibility to utilize various increments of 8 wells per transformation. The wells of the plate are heat sealed, preventing detachment of the aluminum seal in the -80 degrees C freezer and preventing contamination. This format is compatible with automated liquid handling platforms and is intended for medium- to high-throughput use.

The MultiShot format is supplied as pre-aliquoted competent cells in 5, 96-well thin-wall polycarbonate microplates. Each well of the 96-well microplate contains 15 µL of chemically competent cells. This format is compatible with automated liquid handling platforms and is intended for high-throughput use.

We offer the following E. coli strains in the MultiShot FlexPlate format: TOP10, DH5alpha T1R, DH10B T1R, Mach1 T1R, OmniMAX2 T1R, and Stbl3 chemically competent cells.

We offer the following E. coli strains in the MultiShot StripWell format: TOP10, DH5alpha T1R, Mach1 T1R, Stbl3, and BL21 Star (DE3) chemically competent cells.

The MultiShot FlexPlate is a PCR plate and can be placed in a thermal cycler or heat block set at 42 degrees C to transform the cells by the heat shock method. The duration of the heat shock performed in these devices has yielded similar transformation efficiencies as that seen with heat shock done in a water bath.

There are situations where either because of time or process limitations, elimination of the heat shock and recovery time post-heat shock in the transformation protocol would be desirable. Testing was performed on many of our chemically competent cells in both the MultiShot StripWell and MultiShot FlexPlate format with a shortened transformation protocol. Use of a rapid transformation protocol (add DNA, no heat shock, no recovery period, plate on warm agar) yielded up to a log lower in transformation efficiency using the control DNA. This rapid transformation protocol can be performed with expectation of lower colony counts than our standard high efficiency transformation protocol and can only be performed with plasmids that use ampicillin as their antibiotic resistance. The rapid protocol is ideal when using a liquid handling system for high-throughput transformations with high cloning efficiency. Please see the product manual for full details on this rapid transformation protocol option.