JM109 (DE3) 感受态细胞(化转)/ JM109 (

JM109(DE3)chemically Competent Cells

 

JM109(DE3)chemically Competent Cells Components

JM109(DE3)锛?nbsp;                                   100μl/tube

pUC19 (control vector锛?0pg/μl):                    10μl

Storage at                             -80鈩冿紙6month锛?/span>

 

JM109(DE3)chemically Competent Cells Genotype

endA1 recA1 gyrA96 thi-1 hsdR17 (rk-,mk+) relA1 supE44 D (lac-proAB) [F峥?traD36 proAB laqI qZΔM15](DE3)

 

JM109(DE3)chemically Competent Cells Description

JM109 (DE3) is derived from JM109 and is used to efficiently express the gene of the expression vector containing the phage T7 promoter, such as the pET series. The lambda phage DE3 region contains the T7 phage RNA polymerase, which is integrated on the chromosomes of JM109, so it is called JM109 (DE3). T7 RNA polymerase and Escherichia coli RNA polymerase can be expressed at the same time for the protein expression of pET, pGEX, pMAL and other plasmids. The strain of JM109 (DE3) can not be used for the screening test of blue and white. The JM109 (DE3) receptive cells produced by the land - only biological production were made by special technology, and the conversion efficiency of pUC19 plasmid detection was higher than 108 CFU / mu g DNA.

 

JM109(DE3)chemically Competent Cells Transformation Protocol
1. Please store JM109(DE3)chemically Competent Cells in -80°C for late use.
Take it then insert the tube in the ice immediately for 5 minutes.
2. Add your plasmid DNA (vector) to the cell mixture. Carefully flick the tube 4–5 times to mix cells and DNA. Do not vortex.Place the mixture on ice for 25 minutes,Do not mix.
3. Place it in the 42°C water for 45 seconds,then immediately transfer the tube back into ice for 2min. Do not mix.
4. Add 700ul LB or 2YT liquid nutrient medium (No antibiotic). then put on the Constant temperature shaker (200 rpm) for 1 hours at 37°C.
5.Pellet the mixture by centrifugation at 5000rpm/min for 1min. Pour off about 100ul of cell supernatant, then resuspend the cell pellet in the remaining medium by gently vortexing the tube. Spread the cell on the LB or 2YT Resistance plates by antibiotic, then inverted and Incubate overnight at 37°C.

 

Sample Induction Protocol (for reference only)

1. Pick 3-4 transformants for overnight culture in 5 mL LB medium containing antibiotic to select for your expression plasmid. Grow overnight at 37°C with shaking until the OD600 reaches 0.6-1.0.

2.Use the overnight cultures to inoculate fresh LB medium containing antibiotic to an OD600 of 0.05-0.1 (~1:20 dilution of the overnight culture). This dilution allows the cells to quickly return to logarithmic growth and reach the appropriate cell density. Use a volume appropriate for taking time points, if desired.

3.Use the remainder of each overnight culture to create glycerol stocks. Once you have identified the clone that best expresses your protein, you can use the glycerol stock to perform additional expression experiments.

4.Grow the cultures until they reach mid-log phase (OD600 ~0.4; 2 to 3 hours).

5.Induce the cultures (see below), and culture for an additional 2-3 hours. You may also take time points to analyze for optimal expression of your protein.

For T7 expression vector containing the lacI gene (e.g. Invitrogen’s pET vectors), induce by adding L-arabinose to a final concentration of 0.2% AND IPTG to a final concentration of 1 mM.

For T7 expression vector with no lacI gene (e.g. Invitrogen’s pCR®T7 vectors), induce by adding L-arabinose to a final concentration of 0.2%. Culture for an additional 2-3 hours.

6.Place the culture on ice for 10 minutes. Harvest cells by centrifugation at 5,000×g for 10 minutes at 4鈩?

7. Remove the supernatant and store the cell pellet at -20鈩?(storage at lower temperatures is also acceptable). 

 

Caution

1., The competent cells are best to melt slowly in the ice, and insert the target DNA into the ice for 8 minutes. They cannot be placed in the ice for too long. Long time storage will reduce the conversion efficiency.

2. Should be softly manipulate when mixed with plasmids.

3. Transformation of high concentration plasmids could reduce the amount of bacteria that were eventually used for coated plates.

4. When induction, the concentration of IPTG is optional (0.1-2mM).

5. In order to obtain the required amount of protein, the optimum induction time, temperature, and IPTG concentration should be optimized.