Competent Cells Support - Getting Started
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Most of our E. coli strains are K12-derived, and are therefore considered to be non-pathogenic in humans or animals. The exceptions are the BL21 strains (derived from E. coli B which is also considered to be non-pathogenic), Mach1 (derived from E. coli W), and HB101. HB101 is derived from a K12/E. coli B hybrid. Here is the Statement of Non-pathogenicity.
Table of cap color/strain
|Strain||Type||Tube size||Cap color|
|Chem. One Shot|
|ME DH5αT1||One Shot||2.0ml||Yellow|
|ME DH10B T1||One Shot||2.0ml||Green|
|BL21 (DE3)||One Shot||2.0ml||Brown|
|BL21 Star (DE3)||One Shot||2.0ml||Red|
|BL21 Star (DE3)pLysS||One Shot||2.0ml||Blue|
|Chem. Comp. rxns per tube|
|Electrocomp – MultiRxn|
|EM DH10BT1 R||Multireactions||1.5ml||Orange|
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.
|Genotype||gal+, lon-, lac-||gal-, lon+, f80lacZDM15|
|Blue white screening||No||Yes|
|Competency||Chemically competent, >1 x 10e9||ElectroMAX™ electrocompetent >5 x 10e9|
|Packaging||5 x 0.2 mL||5 x 0.1 mL|
Please use our ccdB Survival™ 2 T1R competent cell strain (Cat. No. A10460).
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.
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.
Some suggestions that will help you to obtain the highest transformation efficiency are:
- Thaw competent cells on ice instead of room temperature; do not vortex cells.
- Add DNA to competent cells once thawed.
- Ensure that the incubation times are followed as outlined in the competent cell protocol for the strain you are working with; changes in the length of time can decrease efficiency.
- Remove salts and other contaminants from your DNA sample; DNA can be purified before transformation using a spin column, or phenol/chloroform extraction and ethanol precipitation can be employed.
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:
- Pick one colony into 5 mL LB broth or S.O.C. medium. Grow overnight at 37 degrees C.
- Prepare glycerol solution: 6 mL of S.O.B. medium and 4 mL of glycerol.
- Take one volume of cells and add one volume of glycerol solution and mix.
- Freeze in ethanol/dry ice. Store at –70 degrees C.
S.O.B. Medium (per liter)
|Tryptone (SELECT Peptone 140)||20 g||2%|
|Yeast extract (SELECT Yeast Extract)||5 g||0.5%|
|1 M NaCl||10 mL||10 mM|
|1 M KCl||2.5 mL||2.5 mM|
|1 M MgCl2 (prepared using MgCl2/6H2O, filter-sterilized)||10 mL||10 mM|
|1 M MgSO4 (prepared using MgSO4/7H2O, filter-sterilized)||10 mL||10 mM|
|Distilled water||To a final volume of 1 L|
|Procedure: Add tryptone, yeast extract, NaCl, and KCl to 980 mL of distilled water. Stir to dissolve, autoclave, and cool to room temperature. Add 10 mL of each 1 M magnesium solution.|
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
- Use pUC or pUC-based vectors that contain the portion of the lacZ gene that allows for α complementation.
- Select an E. coli strain that carries the lacZdeltaM15 marker.
- 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.
- 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.
- 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.
You will need bacterial growth media, such as LB, the bacterial selection antibiotic, and induction reagent (IPTG, X-gal). We offer imMedia™ growth medium which is a pre-mixed, pre-sterilized medium containing selection antibiotics for growth of E. coli strains. It can be prepared with or without IPTG and X-gal. Learn more about imMedia™ growth medium here.
We recommend storing our competent E. coli strains at -80◦C. Storage at warmer temperatures, even for a brief period of time, will significantly decrease transformation efficiency.
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.
For Research Use Only. Not for use in diagnostic procedures.