What are XL1-Blue competent cells?
XL-1 Blue Competent Cells for routine cloning allow blue-white color screening, single-strand rescue of phagemid DNA, and preparation of high-quality plasmid DNA. Derivatives enable higher transformation efficiency, transformation of methylated DNA, choice of antibiotic resistance, and no F episome.
How do I make XL1-Blue competent cells?
- Autoclave 100 ml LB- broth in a 1000 ml flask.
- Sterilize and pre-chill the 50-mL conical tubes.
- Prepare 30 ml of ice cold MgCl2-CaCl2 solution (80mM MgCl2, 20 mM CaCl2)
- Prepare ice cold 0.1 CaCl2.
- Pre-warm the LB plates.
How do competent cells make CaCl2?
Resuspend the cells in 1/4 volume of ice cold 0.1 M CaCl2, and leave them on ice for at least 1 hour. To obtain optimal transforming frequency, the cells should be incubated on ice at 4°C for 12-16 hours. Failure to do so will result in a 2-5 fold reduction in transformation frequency.
How is competent cell efficiency calculated?
Transformation efficiency is the efficiency by which cells can take up extracellular DNA and express genes encoded by it. This is based on the competence of the cells. It can be calculated by dividing the number of successful transformants by the amount of DNA used during a transformation procedure.
How are competent cells prepared by the action of CaCl2 Class 12?
CaCl2 is known to increase the efficiency of DNA uptake to produce transformed bacterial cells. The divalent Ca2+ ions supposedly create transient pores on the bacterial cell wall by which the entry of foreign DNA is facilitated into the bacterial cells.
How big is XL1 blue competent cell buffer?
XL1-Blue competent cells (blue tubes) 5 × 0.2 ml ≥1 × 108 pUC18 control plasmid (0.1 ng/µl in TE buffer) 10 µl — β-Mercaptoethanol (1.42 M) 25 µl —. Storage: Supercompetent cells must be placed immediately at the bottom of a –80°C freezer directly from the dry ice shipping container.
When to use XL1-Blue Mr supercompetent cells?
When the F’ episome and blue-white screening are unnecessary, use XL1-Blue MR supercompetent cells. When cloning tetracycline-resistant plasmids, use XL1-Blue MRF’ Kan supercompetent cells to select for the F’ episome and provide a more intense blue color for blue-white screening.
Why are XL1 blue cells recombinase deficient?
XL-1 cells are tetracycline resistant. XL1-Blue cells are endonuclease (endA) deficient, which greatly improves the quality of miniprep DNA, and are recombination (recA) deficient, improving insert stability. The hsdR mutation prevents the cleavage of cloned DNA by the. EcoK endonuclease system.
What is transformation efficiency of XL1 Blue Mr?
Transformation efficiency is ≥1 x 109 transformants/µg of pUC18 DNA. Cells are resistant to kanamycin, and allow blue-white screening for recombinant plasmids. The XL1-Blue MR (Minus Restriction) strain is a restriction minus derivative, useful for cosmid-based cloning.