Invitrogen™ One Shot™ TOP10 Chemically Competent E. coli

Catalog No.	Quantity
C404006	42 x 50 μL
C404010	11 x 50 μL
C404003	21 x 50 μL

Catalog No. C404006 Supplier Invitrogen™ Supplier No. C404006

Description

One Shot TOP10 Chemically Competent E. coli are ideal for high-efficiency cloning and plasmid DNA propagation and are provided at a transformation efficiency of 1 x 10⁹ cfu/μg plasmid DNA.

One Shot TOP10 Chemically Competent E. coli are ideal for high-efficiency cloning and plasmid DNA propagation and are provided at a transformation efficiency of 1 x 10⁹ cfu/μg plasmid DNA. These cells allow stable replication of high-copy number plasmids and are the same competent cells that come with many of our cloning kits. TOP10 E. coli cells are genetically similar to the DH10B strain and have been reported to be more resilient to stress conditions like osmotic shock and acidic pH stress.

One Shot TOP10 cells:
• Maximize cloning efficiency in a single-tube format
• Provide enhanced genomic DNA cloning capabilities

TOP10 Chemically Competent E. coli cells carry mutations in the methylation-dependent restriction system (mcrA, mcrBC, and mrr) allowing the cloning of both prokaryotic and eukaryotic genomic DNA, as well as efficient plasmid rescue from eukaryotic genomes. Similar to other DH strains, TOP10 has the lacZΔM15 genotype, providing for the option of blue-white screening on plates containing either X-Gal or Bluo-Gal. The inclusion of recA1 and endA1 mutations increase insert stability and improve the quality of plasmid DNA prepared from minipreps.

One Shot TOP10 Chemically Competent E. coli cells offer:
• Transformation efficiencies of >1 x 10⁹ cfu/μg
• hsdR for efficient transformation of unmethylated DNA from PCR amplifications
• mcrA for efficient transformation of methylated DNA from genomic preparations
• lacZΔM15 for blue/white color screening of recombinant clones
• endA1 for cleaner DNA preparations and better results in downstream applications due to elimination of nonspecific digestion by Endonuclease I
• recA1 for reduced occurrence of nonspecific recombination in cloned DNA
• Expression from the lac promoter without IPTG

Easy-to-use One Shot format
TOP10 Chemically Competent E. coli cells are supplied in the convenient, single-reaction One Shot format. The single-tube, single-use format accomodates allows all steps of the transformation protocol, up to plating, to take place in the same tube, helping save time and prevent contamination.

Genotype
F^–mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(ara-leu)7697 galU galK λ–rpsL(Str^R) endA1 nupG

Find the strain and format that fit your needs 
• We offer other DH strains in chemically competent and electrocompetent cell formats.
• The TOP10 strain is available in several MultiShot formats for high throughput applications.

Order Info

Shipping Condition: Dry Ice

Specifications

• Product Type: Chemically Competent Cells
• Contains F' Episome: No
• Improves Plasmid Quality: Yes (endA1)
• Cloning Methylated DNA: Yes (mcrA)
• Transformation Efficiency Level: High Efficiency (>1 x 10⁹ cfu/μg)
• Content And Storage: • One Shot TOP10 Chemically Competent E. coli (42 x 50 μL)
  Store Competent Cells at –80°C.
  
  • pUC19 DNA (50 μL at 10 pg/μL)
  Store pUC19 DNA at –20°C.
  
  • S.O.C. medium (6 mL)
  Store S.O.C. Medium at 4°C or room temperature.
  
  
• Antibiotic Resistance Bacterial: Yes (Streptomycin)
• Cloning Unstable DNA: No
• Blue/White Screening: Yes (lacZΔM15)
• High-throughput Compatibility: Low
• Plasmid: High Copy Plasmid
• Preparing Unmethylated DNA: No
• Reduces Recombination: Yes (recA1)
• Shipping Condition: Dry Ice
• T1 Phage - Resistant (tonA): No
• Species: E. coli (K12)
• Format: Tube
• Product Line: One Shot
• Quantity: 42 x 50 μL

Frequently Asked Questions (FAQs)

What is the difference between TOP10 and TOP10F' cells?

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.

I am trying to clone an insert that is supposedly pretty toxic. I used DH5? and TOP10 cells for the transformation and got no colonies on the plate. Do you have any suggestions for me?

If the insert is potentially toxic to the host cells, here are some suggestions that you can try:

- After transforming TOP10 or DH5? cells, incubate at 25-30°C instead of 37°C. This will slow down the growth and will increase the chances of cloning a potentially toxic insert.
- Try using TOP10F' cells for the transformation, but do not add IPTG to the plates. These cells carry the lacIq repressor that represses expression from the lac promoter and so allows cloning of toxic genes. Keep in mind that in the absence of IPTG, blue-white screening cannot be performed.
- Try using Stbl2 cells for the transformation.

Can I directly clone, propagate and express in BL21 without using TOP10?

It is imperative that a cloning strain such as TOP10 be used for characterization of the plasmid, propagation, and maintenance. BL21 cells are wild-type for endA and recA, which could result in poor miniprep quality and a greater chance of plasmid rearrangements due to recombination. In addition, BL21 cells contain the T7 RNA polymerase gene which is expressed at low levels even in the absence of inducer. If the gene is toxic to E. coli, plasmid instability and/or cell death can result.

I need to clone unmethylated DNA from a PCR reaction using a strain that has the hsdRMS mutation to avoid restriction after transformation. Is TOP10 suitable for my purposes?

Yes, TOP10 has the hsdRMS mutation, so this strain can be used to clone DNA from PCR reactions and other non-methylated sources. hsdRMS is a mutation in the system that E. coli uses to recognize foreign DNA. There are two parts to this system, methylation and restriction. E. coli methylate DNA at certain sequences, and if the DNA is not methylated at these sequences it will be recognized as foreign and restricted. Thus, if unmethylated DNA is transformed into E.coli that does not carry the hsdRMS genotype, it is recognized as foreign and enzymatically degraded.

Are TOP10 cells lacIq+ (plus) or lacIq- (minus)? That is, do they produce the lambda lacIq repressor protein?

TOP10 cells are lacIq- (minus). They do not have the lacIq gene and therefore do not produce the lacIq repressor protein. lacIq is most commonly found on an F' episome, and therefore is present in TOP10F', JM101, JM109, and NM522 strains.

Why is Beta-mercaptoethanol (BME) no longer included with the One Shot Chemically Competent E. coli kits? What was the purpose of the BME during E. coli transformation?

Beta-mercaptoethanol (BME) degrades carbohydrates on the cell surface, which theoretically allows DNA to get closer to the membrane prior to heat shock. In the past, this was thought to improve the efficiency of transforming E. coli strains, and the addition of Beta-mercaptoethanol was a standard practice for all chemical transformations. However, galU and galK minus strains, such as TOP10, INV alpha F', DH5 alpha, DH10B and TOP10F', have fewer carbohydrates on the cell surface. After repeated testing of all of our strains, we determined that adding BME had no beneficial effect on transformation efficiency, and we chose to remove BME from the chemically competent One Shot kits.

Is the growth rate of TOP10 cells affected when harboring pZErO-1 plasmids?

Yes, the growth rate of TOP10 cells harboring pZErO-1 will be altered, and depends on how much functional ccdB protein is present. An insert fragment that does not completely disrupt the expression of the LacZ/ccdB fusion (usually very small inserts) will allow some production of the lethal protein which in turn will reduce the growth rate of the cell and produce lower plasmid yields. In contrast, a completely disrupted LacZ/ccdB fusion will allow normal (pUC level) growth and plasmid yield. Typical plasmid yields of pZErO-1 in LB/Zeocin media are 25% - 75% of pUC grown in LB/Amp. Colonies grown in SOB/Zeocin are more healthy and users can expect 75% - 200% of plasmid yield when compared to pUC grown in LB/Amp.

Are your E. coli strains derived from K12?

Most of our E. coli strains are K12-derived. The exceptions are the BL21 strains (derived from E. coli B), Mach1 (derived from E. coli W), and HB101. HB101 is derived from a K12/E. coli B hybrid - See FOCUS, 11:3, p. 56.

I found competent cell vials in my freezer with no box - how can I tell what strain/product it is?

Almost all Invitrogen competent cell vials are labeled by a laser with the strain name and a batch number. The label is etched into the plastic on the side of the vial, but it may be obscured from view by frost in the freezer.

The cap color can also be used to distinguish between products. Below is a list of cap colors for some of our products.

Chemically competent cells cap colors:
TOP10 One Shot - Purple; TOP10F' One Shot - Blue; One Shot Mach1 T1 Phage Resistant - Blue; One Shot OmniMAX2 T1 Phage Resistant - Pink; MAX Efficiency DH5? - Brown; Library Efficiency DH5? - Blue; Subcloning Efficiency DH5? - Clear; One Shot MAX Efficiency DH5?-T1 Phage Resistant - Yellow; One Shot DH10B T1 Phage Resistant - Green; INV?F' One Shot - Clear; MAX Efficiency Stbl2 - Green; One Shot Stbl3 - Clear; INV110 One Shot - Red; BL21 Star(DE3) - Red; BL21 Star(DE3)pLysS - Blue; BL21-AI - Orange; BL21(DE3)pLysE - Pink; BL21(DE3)pLysS - Green; BL21(DE3) - Brown

Electrocompetent cells cap colors:
TOP10 Electrocomp - Yellow; TOP10F' Electrocomp - Green; ElectroMAX DH10B - Yellow; ElectroMAX DH10B T1 Phage Resistant - Orange; ElectroMAX DH5?-E - Red; ElectroMAX Stbl4 - Clear

Safety and Handling

WARNING: Reproductive Harm - www.P65Warnings.ca.gov

For Research Use Only. Not for use in diagnostic procedures.