One Shot™ TOP10/P3 Chemically Competent E. coli
One Shot&trade; TOP10/P3 Chemically Competent <i>E. coli</i>
Invitrogen™

One Shot™ TOP10/P3 Chemically Competent E. coli

One Shot TOP10/P3 Chemically Competent E. coli cells are designed for vector transformation encoding the synthetic supF gene (tyrosine tRNARead more
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Catalog number C505003
Price (USD)
552.00
Each
Add to cart
Price (USD)
552.00
Each
Add to cart

One Shot TOP10/P3 Chemically Competent E. coli cells are designed for vector transformation encoding the synthetic supF gene (tyrosine tRNA suppressor) and allow high-quality pCDM8, pcDNA1.1 plasmid, or any other supF-containing vector preparation.

One Shot TOP10/P3 strain features
• P3 plasmid for selection of supF-containing plasmids
• Mutated endA1 gene for increased quality of plasmid DNA preparations
• recA1 for reduced occurrence of non-specific recombination in cloned DNA
• lacZΔM15 for blue/white color screening of recombinant clones

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 is used to select for cells carrying P3. 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.

Genotype
F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(ara-leu)7697 galU galK rpsL endA1 nupG P3: KanR AmpR (am) TetR (am) 

Find the strain and format that you need
TOP10 is offered in chemically competent and electrocompetent cell formats.
Choose MultiShot competent cells for high-throughput cloning applications.
Email us for more information about custom formats.

For Research Use Only. Not for use in diagnostic procedures.
Specifications
Product TypeChemically Competent Cells
Contains F' EpisomeNo
Improves Plasmid QualityYes (endA1)
Cloning Methylated DNAYes (mcrA)
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 ScreeningYes (lacZΔM15)
High-throughput CompatibilityLow
Preparing Unmethylated DNANo
Reduces RecombinationYes (recA1)
Shipping ConditionDry Ice
T1 Phage - Resistant (tonA)No
SpeciesE. coli (K12)
FormatTube
Product LineOne Shot™
Quantity21 x 50 μL
Unit SizeEach
Contents & Storage
• One Shot TOP10/P3 Chemically Competent E. coli (21 x 50 μL)
Store Competent Cells at –80°C.

• pUC19 Plasmid (50 μL)
Store pUC19 plasmid at –20°C.

S.O.C. Medium (6 mL)
Store S.O.C. Medium at 4°C or at 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.

What is the reason for the nupG mutation in the genotypes for TOP10- and DH10B-related E. coli strains?

nupG is a mutation for the transport of nucleosides. The nupG site is next to endA on the chromosome, and when endA was mutated by transposon insertion, the nupG site was unintentionally mutated as well. There are no apparent effects of this mutation on cell function or growth.

References: 1) Nghiem, Y. et al. PNAS 85: 2709-2713. 2) Westh Hansen, S.V. et al. Eur. J. Biochem. 168: 385-391.

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.

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.