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lleles High Efficiency BL21 (DE3) Competent Cells allow high-efficiency expression of nontoxic proteins in T7 RNA Polymerase-based system. The BL21 (DE3) strain carries a chromosomal copy of the T7 RNA polymerase gene that is controlled by the IPTG inducible lacUV5 promoter.
Box 2 | Storage
Store at -80C It has been observed that long term storage of competent cells at -80C can actually cause the cells to adapt by increasing the recombination rate. Therefore, Allele prepares its competent cells frequently at small scales. It is recommended that competent cells be used within 3 months after arrival.
igh Efficiency BL21 (DE3) Competent Cells offer high quality and efficient protein expression. They are suitable Note: Do not store the competent cells in liquid nitrogen. for a quick 3 min protocol, saving time for researchers. When compared to other leading suppliers Alleles BL21 Competent Cells yield higher colony counts especially for Protocol transformation and ligation reactions. These competent cells are conveniently packaged in 50l aliquots to eliminate For each transformation, thaw one tube of pre-aliquoted the need for repeated freezing and thawing. cells on ice.
Note that it is possible to use 50 l for several transformations. However, if a lower, insufficient volume of competent cells is used, decreased transformation efficiency may occur. 1. Add 0.1100 ng plasmid DNA to each transformation reaction and swirl gently, incubate on ice for 1-2 min. Note: Use more than 10 ng if a Quick transformation protocol is intended (see below). 2. Heat shock in 42C water bath for 45 sec. 3. After heat-shock, incubate the reaction on ice for 1 min. Quick transformation, spread on plate directly.
Genotype
Escherichia coli strain E. coli B F dcm ompT hsdS(rB mB) gal (DE3)
4. Add 900ul of preheated (42C) SOC medium to each transformation reaction and incubate the reactions at 37C for 0.5- 1 hour with shaking at 225250 rpm. 5. Spread 50- 200 l of the cells onto plates that contain the appropriate antibiotics. Induction of target protein using IPTG: 1. Culture Broths: Highly rich culture broths such as Terrific Broth and 2 X YT Broth may support higher expression than LB media. 2. Starting point: IPTG induction between OD600 0.4~1.0. 3. IPTG Concentration: Optimize between 0.01~1 mM 4. Induction Temperature: 37C, 30C, or 15-25C. It depends on your expression system and the recombinant protein. Lower temperature typically helps protein folding. 5. Induction Time: Depending on protein, induce expression to mid-to-late log phase.
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