GB5-alpha™ Electrocompetent E. coli Cells

GoldBio’s GB5-alpha™ Electrocompetent E. coli cells are equivalent to DH5-alpha cells. GB5-alpha™ cells are high efficiency and suitable for a wide variety of applications such as cloning and sub-cloning. Plasmid quality and yields are both enhanced in our GB5-alpha™ cells due to mutations in both endA1 and recA1.

GB5-alpha™ Electrocompetent E. coli cells are equivalent to DH5-alpha.

Reagents Needed for One Reaction

• GB5-alpha™ Electrocompetent cells: 25 µL
• DNA (or pUC19 Control, 500 pg/µL): 0.2 µL
• Recovery medium: 1 mL

Kit Components

• Competent Cells
• 1 x 12 mL Recovery Media
• 1 x 10 µL Control Plasmid (pUC19 Control, 500 pg/µL)

Storage/Handling

This product may be shipped on dry ice. GB5-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

• ≥5 x 10⁸ cfu/µg efficiency with electroporation
• Enhanced plasmid yield and quality
• Blue/white screening
• Enhanced insert stability

Genotype

F^– φ80lacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(r_K^–, m_K⁺) phoA supE44 λ^–thi-1 gyrA96 relA1

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 ≥5 x 10⁸ 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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