BL21Condon Plus (DE3)-RIPL 感受态

BL21CONDON PLUS (DE3)-RIPL Chemically Competent Cell

Packing 10 x 0.1 ml/tube

Store at –80°C

Genotype
E. coli B F- ompT hsdS(rB- mB-) dcm+ Tetr gal λ(DE3) endA Hte [argU proL Camr]  [argU ileY leuW Strep/Specr]

Alternative name
BL21CONDON PLUS (DE3)-RIPL chemically Competent Cells,BL21CONDON PLUS (DE3)-RIPL  Competent E.coli,BL21CONDON PLUS (DE3)-RIPL Competent Cells, BL21CONDON PLUS (DE3)-RIPL chemically E.coli Express Competent Cells

Description

      The original K-12 wild-type strain from the Stanford collection contained both the F plasmid and phage lambda. MG1655 was constructed by curing that strain using acridine orange and UV.This strain was sequenced by the Blattner laboratory because it approximates wild-type E. coli and "has been maintained as a laboratory strain with minimal genetic manipulation, having only been cured of the temperate bacteriophage lambda and F plasmid by means of ultraviolet light and acridine orange, respectively." (Blattner, et al. 1997). The mutations listed in the genotype are present in most K-12 strains and were probably acquired early in the history of the laboratory strain. A frameshift at the end of rph results in decreased pyrE expression and a mild pyrimidine starvation, such that the strain grows 10 to 15% more slowly in pyrimidine-free medium than in medium containing uracil (Jensen 1993). The ilvG- mutation is a frameshift that knocks out acetohydroxy acid synthase II (Lawther, et al. 1982). The rfb-50 mutation is an IS5 insertion that results in the absence of O-antigen synthesis (Liu and Reeves 1994).
          MG1655 was derived and named by Mark Guyer from strain W1485, which was derived in Joshua Lederberg's lab from a stab-culture descendant of the original K-12 isolate. This original E. coli strain K-12 was obtained from a stool sample of a diphtheria patient in Palo Alto, CA in 1922 (Bachmann, B., pp. 2460-2488 in Neidhardt et al.1996, Escherichia coli and Salmonella: Cellular and Molecular Biology, ASM Press).MG1655 grows on LB and M9 minimal medium (+ Glucose + 1ug/ml thiamine).

Transformation Protocol

1.Please store BL21CONDON PLUS (DE3)-RIPL  Chemically Competent Cell in -80°C for late use.

2. Thaw a tube of BL21CONDON PLUS (DE3)-RIPL  Chemically Competent Cell锛?00μl 锛塷n ice.

3. Add 1µl plasmid DNA or 10µl ligation product to the cell mixture. Carefully flick the tube 4–5 times to mix cells and DNA. Do not vortex.

4.Place the mixture on ice for 30 minutes. Do not mix.

5. Heat shock at exactly 42°C for exactly 90 seconds and then immediately transfer the tube back into ice for 5min. Do not mix.

6. Place on ice for 5 minutes. Do not mix.

7. Add 800ul LB then put on the Constant temperature shaker (150 rpm) for 45min,Warm selection plates to 37°C.

8. Pellet the mixture by centrifugation at 4000rpm/min for 3min. Pour off about 200ul of cell supernatant, then resuspend the cell pellet in the remaining medium by gently vortexing the tube.

9.Spread the cell on the agar plates by antibiotic, then place it on 37°C for 1hour.

10. Incubate overnight at 37°C.

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!