E. coli Calcium Chloride competent cell protocol
1）Streak E.coli cells (DH5a) on an LB plate; (BL21(DE3)LysS cells on LB plate+34 mg/ml chloramphenicol)
2） Allow cells to grow at 37℃ overnight
3）Place one colony in 10 ml LB media (+antibiotic selection if necessary), grow overnight at 37℃
4） Take 2 ml LB media and save for blank. Transfer 5 ml overnight DH5a culture into 500 ml LB media in 3 L flask
5） Allow cell to grow at 37℃ (250 rpm), until OD600= 0.4 (~2-3 hours)
6） Transfer cells to 2 centrifuge bottles (250 ml), and place cells on ice for 20 min
7） Centrifuge cells in at 4oC for 10 min at 3,000 g and subsequent resuspension may be done in the same bottle. Cells must remain cold for the rest of the procedure: Transport tubes on ice and resuspend on ice in the cold room
8） Pour off media and resuspend cells in 30 ml of cold 0.1 M CaCl2. Transfer the suspended cells into 50 ml polypropylene tubes, and incubate on ice for 30 min
9） Centrifuge cells at 4O℃ for 10 min at 3,000 g
10） Pour supernatant and resuspend cells (by pipetting) in 8 ml cold 0.1M CaCl2 containing 15% glycerol. Transfer 140 ml into (1.5 ml) Ependorff tubes placed on ice. Freeze the cells in liquid nitrogen. Cells stored at -80oC can be used for transformation for up to ~6 months
11） Add 10 to 40 ng (10 to 25 ml volume) of DNA to 250 ml of competent cells in step
12） Incubate the mixture on ice for 30 minutes.
13） Transfer the reaction to a 42℃ water for 1min.
14） Add 0.9 ml of LB culture to each tube and incubate at 37℃ for 1 hour in a roller drum (250 rpm) to allow cells to recover and express the antibiotic resistance marker.
15） Incubate on ice for 2 minutes.
16） Spread the appropriate quantity of cells (50 to 100 ml) on selective media. Store the remaining cells at 4℃.
(A) E. coli cells from the control tube without DNA in step 12 above are plated on selective medium and nonselective medium. The first plating ensures that the selective medium is working properly since no growth should be observed. The second plating provides the number of viable cells in the absence of selective medium.
(B) E. coli cells being tested for competency are plated on LB agar containing ampicillin (50 mg/ml final concentration) to ensure that the transformation efficiency has not decreased over time due to storage.
17） Incubate all plates overnight at 37℃ (agar side up).
18） Count the number of colonies.
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