MC1061/P3 Chemically Competent E. coli
MC1061/P3 Chemically Competent <i>E. coli</i>
Invitrogen™

MC1061/P3 Chemically Competent E. coli

MC1061/P3 Chemically Competent E. coli are used for highly efficient transformations that require the P3 episome for selection and maintenanceRead more
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Catalog number C66303
Price (USD)
397.00
Each
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Price (USD)
397.00
Each
Add to cart

MC1061/P3 Chemically Competent E. coli are used for highly efficient transformations that require the P3 episome for selection and maintenance of vectors encoding the tyrosine tRNA suppressor (synthetic supF gene, such as pCDM8, pcDNA1.1, or any other supF-containing vector).

MC1061 E. coli cells were used by Hanahan and coworkers to engineer the DH10B strain. Among the replaced genes by adding mutations were recA1 (inhibiting the homologous recombination system), endA1, and lacZΔM15. Thus, this strain with its intact homologous recombination system is used in applications where recombination is needed, such as in genetically engineering E. coli by recombination or plasmid-phage recombination.

MC1061/P3 Chemically Competent E. coli offer:
• Transformation efficiencies of >1 x 108 cfu/μg
• P3 plasmid for selection of supF-containing plasmids
• hsdR for efficient transformation of unmethylated DNA from PCR amplifications

P3 plasmid
The P3 plasmid is a low-copy-number, 60 kb plasmid that carries kanamycin, tetracycline, and ampicillin resistance markers. The kanamycin gene is fully active and used to select for cells carrying P3. The tetracycline and ampicillin genes carry amber mutations, which render the genes inactive during normal growth and replication of the bacteria. Upon transformation of a vector carrying the suppressor F gene, the amber mutations in the tetracycline and ampicillin genes on the P3 plasmid are suppressed, and the E. coli are resistant to these antibiotics.

Note: The spontaneous reversion rate for the amber mutation in the ampicillin marker is 5%, while the reversion rate for the amber mutation in the tetracycline marker is 1%.To minimizing the reversion rates, use tetracycline and ampicillin together for selection of E. coli (P3) cells transformed with the SupF plasmid.

Genotype
F–hsdR(rK–, mK+) araD139 Δ(araABC-leu)7679 galU galK ΔlacX74 rpsL(StrR) thi mcrB / P3: KanR AmpR (am) TetR (am)

Find the strain and format that fits your needs
We offer DH strains in chemically competent and electrocompetent cell formats to meet your specific needs.
The TOP10/P3 strain could be used where recombination is not needed.
Strains are available in several MultiShot formats for high throughput applications.
Explore bacterial growth media formats.

For Research Use Only. Not for use in diagnostic procedures.
Specifications
Product TypeChemically Competent Cells
Contains F' EpisomeNo
Improves Plasmid QualityNo
Cloning Methylated DNAYes (hsd)
Transformation Efficiency LevelMedium Efficiency (1 x 108 to 1 x 109 cfu/μg)
Antibiotic Resistance BacterialYes (Streptomycin, Kanamycin, Ampicillin, Tetracycline)
Cloning Unstable DNANo
Blue/White ScreeningNo
High-throughput CompatibilityLow
Preparing Unmethylated DNANo
Reduces RecombinationNo
Shipping ConditionDry Ice
T1 Phage - Resistant (tonA)No
SpeciesE. coli (K12)
FormatTube
Quantity5 x 300 μL
Unit SizeEach
Contents & Storage
• MC1021/P3 Chemically Competent E. coli (5 x 300 μL)
Store Competent Cells at –80°C.

• pUC19 DNA (1 x 50 μL)
Store pUC19 DNA at –20°C.

• S.O.C. Medium (15 ml)
Store S.O.C. Medium at 4°C or room temperature.

Frequently asked questions (FAQs)

What is the mechanism for supF selection in E. coli carrying the P3 plasmid? And what is meant by the ''amber'' notation in the genotype - are these cells resistant to tetracycline and ampicillin?

E. coli harboring the plasmid P3 enable the selection and maintenance of plasmids that encode the tRNA suppressor F gene (supF). P3, a low-copy 60 kb episomal plasmid, encodes the kanamycin resistance gene as well as amber mutants of the tetracyline and ampicillin resistance genes. The amber mutations are point mutations in the resistance markers which inactivate the expression of resistance in the strain unless the tRNA supressor F (supF) is present. Therefore, strains that harbor P3 alone are resistant to kanamycin, but sensitive to both tetracycline and ampicillin. But when the E. coli carrying the P3 plasmid are transformed with supF-containing plasmids (e.g. pcDNA1 and pCDM8), they are rendered resistant to both tetracycline and ampicillin (as well as kanamycin) by suppression of the amber mutations.

The rate of spontaneous reversion of the amber point mutations on the P3 episome is fairly high, so it is important to select supF clones with both tetracycline and ampicillin resistance to reduce background growth. To avoid enriching for revertants, it is recommended that relatively low concentrations of tetracycline (7.5 to 10 ug/ml) and ampicillin (25 to 40 ug/ml) be used.

How large is the P3 plasmid in the MC1061/P3 or TOP10/P3 cells, and is it isolated with other plasmids in standard preps?

The P3 plasmid is approximately 60 kb and usually is present at only 1 or 2 copies per cell. It is similar in properties to the F' episome. It can be recovered along with your vector of interest in a plasmid prep and might be detectable on a gel. The very large plasmid would be seen high on the gel near the wells.

When examining mini-prep DNA isolated from MC1061/P3, there is a smear extending from high molecular weight to ~5 kb instead of an apparent band at or near where the vector should be. What causes this?

The smear is expected when doing DNA preps in MC1061/P3 because the cells do not carry the endA- mutation. To prevent DNA degradation by endonuclease in plasmid preparations from these cells, an alkaline lysis procedure (i.e. Solutions I,II,III) which contains a phenol:chloroform step that destroys endogenous endonucleases should be used. Many commercially available DNA miniprep kits do not accomplish this efficiently, and boiling the DNA solution after isolation is not effective.

For best DNA preparation results with supF-selection plasmids, we highly recommend the TOP10/P3 One Shot cells (catalog number C5050-03). This strain yields higher quality and quantity of plasmid DNA due to the presence of both recA- and endA- mutations. For plasmids with antibiotic selection markers rather than supF selection, use one of the more common strains like TOP10, DH5a or DH10B.

What's the difference between MC1061/P3 and TOP10/P3, and how would I choose one or the other for my supF selection (for example: using pcDNA1 or pcDNA1.1)?

MC1061/P3 is the strain that was historically used for most supF selection applications. It is referenced by Brian Seed in his library construction paper.

For library transformations, we do still recommend MC1061/P3 because of its slightly higher transformation efficiency when transforming pcDNA1.1. However, for propagating plasmid for transfection, we strongly recommend using the TOP10/P3 instead, which is endA- and yields much cleaner plasmid DNA in purification. DNA prepared from MC1061 cells is typically not suitable for transfection.