BL21 (DE3) chemically E.coli Express Competent Cells

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  • Model: CHC00001
  • 100 Units in Stock
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BL21(DE3) chemically Competent Cells

BL21(DE3):                                       10×100μl

pUC19(control vector):  10pg/μl        

Store at –80°C

Genotype  

F- ompT hsdSB(rB- mB- ) gal dcm(DE3)

Alternative name
BL21 (DE3) chemically Competent Cells,BL21 (DE3) Competent E.coli Express Competent Cells, BL21 (DE3) chemically E.coli Express Express Competent Cells 

Transformation Protocol

1.   Inoculate a single colony from a freshly streaked plate into 5 ml of LB medium containing the appropriate antibiotic for the plasmid and host strain.

2.   Incubate with shaking at 200 rpm at 37℃ overnight. 

3.   Inoculate 50 ml of LB medium containing the appropriate antibiotic with 0.5 ml of the overnight culture prepared in step 2(use the 500 ml triangular flask as the container would be better).

4.   Incubate with shaking at 150 rpm at 37℃ until the OD 600 reaches 0.5-0.8.

5.   (Optional)Pipet 1ml of  the cultures into clean microcentrifuge tubes and  place the tubes on ice until  needed  for gel analysis or storage at  -20℃. These will serve as the non-induced control samples.     

6.   Add IPTG to a final concentration of 1 mM.  Optimal time for induction of the target protein may vary from 2-16 hours, depending on the protein.

7.   Incubate with shaking at 120 rpm at 37℃ for 3-4 hours. To determine the optimal time for induction of the target protein, it is recommended that a time course experiment be performed varying the induction from 2-16 hours.

8.   Place the culture on ice for 10 minutes. Harvest cells by centrifugation at 5,000×g for 10 min at 4℃.

9.   Remove the supernatant and store the cell pellet at -20℃ (storage at lower temperatures is also acceptable). 

IPTG

Prepare a 1 M solution of IPTG (Isopropyl-β-D-thiogalactoside; Isopropyl-β-D-thiogalactopyranoside) bydissolving 2.38 g of IPTG in dd water and adjust the final volume to 10 ml. Filter sterilize before use.

Caution:

1.STORAGE AND HANDLING: Competent cells should be stored at

–80°C. Storage at –20°C will result in a significant decrease in transformation efficiency. Cells lose efficiency whenever they are warmed above –80°C, even if they do not thaw.

2. This product is FOR RESEARCH USE ONLY!

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