CJ236 Electrocompetent E. Coli Cells

GoldBio CJ236 Electrocompetent E. coli cells are ideally suited for use in the Kunkel method of mutagenesis. CJ236 cells have part of their Thymine replaced with deoxyuracil in the DNA due to dut- and ung- mutations which prevent the cells from removing uracil from DNA caused by misincorporation or dCTP deamination. CJ236 contains the F’ gene which confers Chloramphenicol resistance.

Kit Components

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

Product Specifications
Competent cell type: Electrocompetent
Derivative of: CJ236
Species: E. coli
Transformation efficiency: ≥1 x 10⁹ cfu/µg pUC19 DNA
Blue/white screening: No

Storage/Handling: This product may be shipped on dry ice. CJ236 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.

Genotype

dut1,ung1,thi-1,recA1 / pCJ105 (F' cam^r)

Reagents Needed for One Reaction

• CJ236 electrocompetent cells: 25 µL
• DNA (or pUC19 Control, 500 pg/µL): 0.2 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 ≥1 x 10⁹ 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 1µg 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