Invitrogen™ Library Efficiency™ DH5α Competent Cells

Catalog No.	Quantity
18-263-012	5 x 200 μL

Catalog No. 18-263-012 Supplier Invitrogen™ Supplier No. 18263012

Description

Includes

Library Efficiency DH5α Competent Cells: 5 vials, 200μL each (total of 1mL), pUC19 DNA (10pg/μL): 1 vial, 50μL, S.O.C. medium: 2 bottles, 6mL each

Library Efficiency DH5α Competent Cells are a versatile strain of chemically competent cells that are used in many everyday cloning applications.

Library Efficiency DH5α Competent Cells are a versatile strain of chemically competent cells that are used in many everyday cloning applications. Prepared by a proprietary modification of the Hanahan method, these cells are able to achieve transformation efficiencies of >1 x 10⁸ cfu/μg control DNA per 100 μL reaction.  
 
Library Efficiency DH5α Competent Cells are recommended for routine cloning of genes into large plasmids and are suitable for difficult clone constructions, such as blunt-end ligations or cloning using limited amount of DNA.

The strain has the lacZΔM15 genotype, allowing blue-white screening on plates containing either X-Gal or Bluo-Gal. It also carries the recA1 mutation, which helps reduce the rate of recombination while propagating plasmid DNA, and the endA1 mutation that makes DH5α cells an excellent choice for amplifying plasmid DNA for subsequent extraction and purification. DH5α cells are capable of being transformed efficiently with large plasmids and can serve as a host for the M13mp cloning vectors if a lawn of DH5αF′IQ (Cat. No. 18288019), for example, is provided to allow plaque formation.

Library Efficiency DH5α Competent Cells offer: 
• Transformation efficiencies of >1 x 10⁸ cfu/μg
• lacZΔM15 marker that provides α-complementation of the β-galactosidase gene allowing blue/white screening on agar plates containing X-Gal or Bluo-Gal
• Plasmid DNA maintained with minimum recombination events due to recA1 mutation
• endA1 mutation that enables increased plasmid yield and quantity
• hsdR17 genotype that allows efficient transformation of cloned unmethylated DNA from PCR

Genotype
F^- Φ80lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17 (rk-, mk+) phoA supE44 λ-thi-1 gyrA96 relA1

Find the strain and format that fits your needs
• DH5α strain and its derivatives are available with a variety of transformation efficiencies and in both electrocompetent and chemically competent formats.
• MAX Efficiency DH5αF´IQ Competent Cells (Cat. No. 18288019) are available in chemically competent format.
• DH5α-T1R competent cells are available in MultiShot format 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: No
• Transformation Efficiency Level: Medium Efficiency (1 x 10⁸ to 1 x 10⁹ cfu/μg)
• Content And Storage: • Library Efficiency DH5α Competent Cells (5 x 200 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 (2 x 6 mL)
  Store S.O.C. Medium at 4°C or room temperature.
  
  
• Antibiotic Resistance Bacterial: No
• Cloning Unstable DNA: Not suitable for cloning unstable DNA
• Blue/White Screening: Yes (lacZΔM15)
• High-throughput Compatibility: Low
• 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: Library Efficiency
• Quantity: 5 x 200 μL

Frequently Asked Questions (FAQs)

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.

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.

You offer competent cells in Subcloning Efficiency, Library Efficiency and MAX Efficiency. How do these differ?

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

Do any Invitrogen competent cells contain DMSO in the freezing medium?

Yes, several of our competent cells products are frozen with DMSO. The presence of DMSO (dimethylsulfoxide) will generally be indicated in the MSDS files if you have a question about a particular product, but here is a list of commonly used products that are known to have DMSO in the freezing buffer:

One Shot OmniMAX 2 T1 Phage Resistant Cells, Cat. No. C8540-03

One Shot INV?F' Chemically Competent Cells, Cat. No. C2020-03 and C2020-06

One Shot MAX Efficiency DH5?-T1 Chemically Competent Cells, Cat. No. 12297-016

MAX Efficiency DH5?-T1 Phage Resistant Cells, Cat. No. 12034-013

MAX Efficiency DH5? Chemically Competent Cells, Cat. No. 18258-012

Library Efficiency DH5? Chemically Competent Cells, Cat. No. 18263-012

MAX Efficiency DH5? F'IQ Cells, Cat. No. 18288-019

MAX Efficiency Stbl2Chemically Competent Cells, Cat. No. 10268-019

Is S.O.C. medium absolutely required when recovering competent bacterial cells during transformation?

Many media can be used to grow transformed cells, including standard LB, SOB or TB broths. However, S.O.C. is the optimal choice for recovery of the cells before plating. The nutrient-rich formula with added glucose is often important for obtaining maximum transformation efficiencies.

Why is it necessary to dilute ligated DNA products before adding them to competent bacterial cells?

Components of the ligation reaction (enzymes, salts) can interfere with transformation, and may reduce the number of recombinant colonies or plaques. We recommend a five-fold dilution of the ligation mix, and adding not more than 1/10 of the diluted volume to the cells. For best results, the volume added should also not exceed 10% of the volume of the competent cells that you are using.

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.