GB5-alpha™ Chemically Competent E. coli Cells

GoldBio’s GB5-alpha™ Chemically Competent E. coli cells are equivalent to DH5-alpha cells and are high efficiency cells suitable for transformation in a wide variety of routine applications such as plasmid isolation, cloning, and subcloning.

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

Reagents Needed for One Reaction

• GB5-alpha™ chemically competent cells: 50 µL
• DNA (or pUC19 Control, 500 pg/µL): 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™ Chemically Competent 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.

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 10⁶ 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.

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