Invitrogen™ One Shot™ ccdB Survival™ 2 T1^R Competent Cells

Product Code.	Quantity	unitSize
10733874	11 x 50 μL	Each

Product Code. 10733874 Supplier Invitrogen™ Supplier No. A10460

Description

Includes

ccdB Survival2 T1R Competent Cells: 11 vials, 50μL each, pUC19 DNA (10pg/μL): 1 vial, 50μL, S.O.C. medium: 1 bottle, 6mL

One Shot ccdB Survival 2 T1^R Competent Cells are suitable for the propagation of plasmids containing the ccdB gene and are designed for use with the Gateway Vector Conversion System and for propagating Gateway destination, donor, and supercoiled entry vectors.

One Shot ccdB Survival 2 T1^R Competent Cells are suitable for the propagation of plasmids containing the ccdB gene and are designed for use with the Gateway Vector Conversion System and for propagating Gateway destination, donor, and supercoiled entry vectors. ccdB Survival 2 T1R cells that are made chemically competent can achieve high transformation efficiency of >1 x 10⁹ transformants/μg plasmid DNA.

The ccdB Survival 2 T1^R E. coli strain is derived from the TOP10 strain and offers the same benefits. Mutations in the methylation-dependent restriction system (mcrA, mcrBC, and mrr) allow cloning of both prokaryotic and eukaryotic genomic DNA that contains methylcytosine and methyladenine. This strain also has the lacZΔM15 genotype, providing the option of blue-white screening on plates containing either X-Gal or Bluo-Gal. In addition, recA1 and endA1 mutations increase insert stability and improve the quality of plasmid DNA prepared from minipreps.The tonA (also known as fhuA) mutation in ccdB Survival 2 T1^R cells confers resistance to bacteriophages T1 and T5, safeguarding samples against contamination.

ccdB Survival 2 T1^R Competent Cells offer:
• >1 x 10⁹ transformants/μg efficiency
• tonA (fhuA) genotype to confer resistance to T1 and T5 phage
• Support of high-yield preparation of supercoiled plasmid DNA (the endA1 phenotype)
• Reduced nonspecific recombination of cloned DNA (the recA1 phenotype) 

Convenient and efficient format
One Shot ccdB Survival 2 T1^R E. coli are supplied in the convenient, single-reaction One Shot format. Each tube contains enough cells for one transformation so there are no efficiency-zapping, freeze-thaw cycles or money wasted on unused cells.

Genotype: F^-mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1 araΔ139 Δ(ara-leu)7697 galU galK rpsL(Str^R) endA1 nupG fhuA::IS2

Find the strain and format that fits your needs
• We offer other DH strains in chemically competent and electrocompetent cell formats to meet your specific needs.
• MultiShot formats for high throughput applications are available.

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 ccdB Survival 2 T1R Competent Cells (11 x 50 μL)
  Store Competent Cells at &ndash80°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: Not suitable for cloning unstable DNA
• Blue/White Screening: Yes (lacZΔM15)
• High-throughput Compatibility: Low
• Plasmid: ccdB vector propagation
• Preparing Unmethylated DNA: No
• Reduces Recombination: Yes (recA1)
• Shipping Condition: Dry Ice
• T1 Phage - Resistant (tonA): Yes
• Species: E. coli (K12)
• Format: Tube
• Product Line: One Shot
• Quantity: 11 x 50 μL

Frequently Asked Questions (FAQs)

I just found out that Library Efficiency DB3.1 and One Shot ccdB Survival T1R cells have been discontinued. Do you offer an alternative for propagating ccdB-containing plasmids?

We offer One Shot ccdB Survival 2 T1^R cells (Cat. No. A10460) for propagating ccdB-containing plasmids.

What kind of competent cells should I use to propagate Donor vectors and Destination vectors? What cells should I use after the BP or LR recombination reaction?

All Donor vectors and Destination vectors contain the ccdB cell death gene to reduce background of non-recombined BP/LR plasmids. Therefore, growing non-recombined vector requires special cells (One Shot ccdB Survival 2 T1R Competent Cells) which are resistant to the lethal effects of ccdB. On the other hand, general E. coli cloning strains including TOP10 or DH5a may be used for plating the BP or LR reaction, or for propagation and maintenance of recombined Gateway constructs.

I need a competent cell strain that is ccdB resistant. The Library Efficiency DB3.1 cells you offer have been discontinued, as well as the One Shot ccdB Survival T1R chemically competent cells. What do you recommend?

Please use our ccdB Survival 2 T1R competent cell strain.

How do you recommend that I prepare my DNA for successful electroporation of E. coli?

For best results, DNA used in electroporation must have a very low ionic strength and a high resistance. A high-salt DNA sample may be purified by either ethanol precipitation or dialysis.

The following suggested protocols are for ligation reactions of 20ul. The volumes may be adjusted to suit the amount being prepared.

Purifying DNA by Precipitation: Add 5 to 10 ug of tRNA to a 20ul ligation reaction. Adjust the solution to 2.5 M in ammonium acetate using a 7.5 M ammonium acetate stock solution. Mix well. Add two volumes of 100 % ethanol. Centrifuge at 12,000 x g for 15 min at 4C. Remove the supernatant with a micropipet. Wash the pellet with 60ul of 70% ethanol. Centrifuge at 12,000 x g for 15 min at room temperature. Remove the supernatant with a micropipet. Air dry the pellet. Resuspend the DNA in 0.5X TE buffer [5 mM Tris-HCl, 0.5 mM EDTA (pH 7.5)] to a concentration of 10 ng/ul of DNA. Use 1 ul per transformation of 20 ul of cell suspension.

Purifying DNA by Microdialysis: Float a Millipore filter, type VS 0.025 um, on a pool of 0.5X TE buffer (or 10% glycerol) in a small plastic container. Place 20ul of the DNA solution as a drop on top of the filter. Incubate at room temperature for several hours. Withdraw the DNA drop from the filter and place it in a polypropylene microcentrifuge tube. Use 1ul of this DNA for each electrotransformation reaction.

When should DMSO, formamide, glycerol and other cosolvents be used in PCR?

Cosolvents may be used when there is a failure of amplification, either because the template contains stable hairpin-loops or the region of amplification is GC-rich. Keep in mind that all of these cosolvents have the effect of lowering enzyme activity, which will decrease amplification yield. For more information see P Landre et al (1995). The use of co-solvents to enhance amplification by the polymerase chain reaction. In: PCR Strategies, edited by MA Innis, DH Gelfand, JJ Sninsky. Academic Press, San Diego, CA, pp. 3-16.

Additionally, when amplifying very long PCR fragments (greater than 5 kb) the use of cosolvents is often recommended to help compensate for the increased melting temperature of these fragments.

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