TFB Blue™- 25 Pack

TFB Blue™ Competent Cells are chemically competent E. coli cells and are intended to be transformed by heat-shock. The high transformation efficiency of these cells make them ideally suited for demanding applications such as cDNA library construction, gene banks and M13 bacteriophage work including the production of single-stranded DNA.

TFB Blue™ cells are resistant to bacteriophage T1, an especially virulent virus which attacks E. coli, and can be rapidly spread via aerosols. The increased amount of Lac repressor in these cells, due to the presence of the lacIq marker, gives tighter control over Lac-based promoter systems making them ideal for cloning and expression of toxic genes (1).

Handling and Storage

• Upon receipt, store cells immediately at -70°C or below.
• Competent cells should be thawed on wet ice (~ 10 minutes) and should be used promptly after thawing.
• Thawed cells can be refrozen by placing them in dry ice although a drop in transformation efficiency of as much as ≥50% may be observed.
• Cells stored properly will remain stable for up to 2 years.

Kit Contents

• TFB Blue™ Competent Cells
• pUC19 Control DNA (10 pg / µl)

Genotype

F’[lacI^q Tn10 (Tet^R)] / Φ80dlacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(r^-_k, m⁺_k) phoA supE44 λ^- thi-1 gyrA96 relA1 fhuA (T1^R). For optimal repression of the lacZ promoter, grow cells at 30°C.

Quality Control

TFB Blue™ cells are tested to verify transformation efficiencies of ≥ 1 x 10⁹ transformants/µg with limiting amounts of supercoiled pUC19 DNA (50 pg) and greater than 1 x 10⁶ cfu’s per 100 µl transformation when using 25 ng of pUC19 (i.e., "saturating amounts"). Untransformed cells are also tested for resistance to bacteriophage T5, a standard test for resistance to phage T1, as well as other relevant genetic markers and sensitivities to antibiotics.

Considerations in Choosing Competent Cells

Key Genetic Markers:

• recA - eliminates homologous recombination; stabilizes inserts.
• endA - endonuclease deficiency improves yield and quality of plasmid DNA.
• hsd - classical restriction/modification system, deficiency allows cloning from non-E. coli sources, e.g. PCR products.
• F’ - episome required for infection by M13 and other phagemids. Important in generation of ssDNA.
• mcrA, mcrBC, mrr - mutations in these genes allows efficient cloning of genomic from mammalian or other higher eucaryotes, and methylated cDNA.
• lacZΔM15 - omega donor; allows a-complementation for blue/white screening.

Product Selection Guide:

• Competent Cells Selection Guide