DH5-alpha Electrocompetent E. coli Cells

GoldBio’s DH5-alpha Electrocompetent E. coli cells are suitable for high efficiency transformation in a wide variety of routine applications such as plasmid isolation, cloning, and subcloning. Mutations in endA1 and recA1 ensure increased plasmid yield and improved plasmid quality.

Product Specifications
Competent cell type: ElectroCompetent
Derivative of: DH5-alpha
Species: E. coli
Format: Tubes
Transformation efficiency: ≥2 x 10¹⁰ cfu/µg pUC19 DNA
Blue/white screening: Yes

Storage/Handling: This product may be shipped on dry ice. DH5-alpha Electrocompetent E. coli cells should be stored at -80°C, pUC19 Control DNA should be stored at -20°C and recovery medium should be stored at 4°C immediately upon arrival. When stored under the recommended conditions and handled correctly, these products should be stable for at least 1 year from the date of receipt.

Genomic Features

• ≥2 x 10¹⁰ cfu/µg efficiency with electroporation
  
• Greatly increased plasmid yield and quality due to endA1 mutation
  
• High-efficiency transformation with plasmids 30 kb in size
  
• Blue/white screening of recombinant clones due to lacZΔM15
  
• Ensured insert stability due to recA1 mutation

Genotype
Φ80 Δ(lacZ)M15 fhuA2 Δ(argF-lacZ)U169 phoA glnV44 gyrA96 recA1 relA1 endA1 thi-1hsdR17

Reagents Needed for One Reaction

• DH5-alpha electrocompetent cells: 25 µl
  
• DNA (or pUC19 Control, 10 pg/µl): 1 µl
  
• Recovery medium: 1 ml

Quality Control
Transformation efficiency is tested by using the pUC19 control DNA supplied with the kit and using the protocol given below. Transformation efficiency should be ≥2 x 10¹⁰ CFU/µg pUC19 DNA. Untransformed cells are tested for appropriate antibiotic sensitivity.

General Guidelines

• Handle competent cells gently as they are highly sensitive to changes in temperature or mechanical lysis caused by pipetting.
  
• Thaw competent cells on ice, and transform cells immediately following thawing. After adding DNA, mix by tapping the tube gently. Do not mix cells by pipetting or vortexing.

Note: A high-voltage electroporation apparatus capable of generating field strengths of 16 kV/cm is required.

Calculation of Transformation Efficiency
Transformation Efficiency (TE) is defined as the number of colony forming units (cfu) produced by transforming 1 µg of plasmid into a given volume of competent cells.

• TE = Colonies/µg/Dilution
  • Colonies = the number of colonies counted
    
  • µg = amount of DNA transformed in µg
    
  • Dilution = total dilution of the DNA before plating

Example: Transform 1 µl of (10 pg/µl) control plasmid into 25 µl of cells, add 975 µl of Recovery Medium. Dilute 10 µl of this in 990 µl of Recovery Medium and plate 50 µl. Count the colonies on the plate the next day. If you count 250 colonies, the TE is calculated as follows:

Colonies = 250
µg of DNA = 0.00001
Dilution = 10/1000 x 50/1000 = 0.0005
TE = 250/0.00001/0.0005 = 5.0 × 10¹⁰

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