Control culture of Escherichia coli, isolation of single colony and preservation of strains

Control culture of Escherichia coli, isolation of single colony and preservation of strains

Escherichia coli is a receptor bacterium widely used in molecular genetics experiments and genetic engineering operations. Its genetic background has been studied in detail, and it is often used as a host for gene amplification and exogenous gene expression.
[Experimental materials]: E. coli InvaF’E. coli InvaF’ (pUC19)
[Experimental Instruments] : Ultra-clean workbench, constant temperature incubator, autoclave, constant temperature water bath, microwave oven, etc.
[Reagents]: Agar, Yeast Extract, Tryptone, NaCl, Ampicillin, NaOH, etc.
[Experimental procedure]
1. Prepare the medium:
a. LB(Lurib-Bertani) media
Yeast extract 5g/L
Tryptone (trytone) 10g/L
NaCl 10g/L
Add distilled water to 1000ml to adjust pH to 7.0.
Preparation of 750ml solid medium: In the above liquid medium, add agar at 15 g/L and heat until fully melted to form a solid medium.
b: 50% glycerol: Take 50ml of glycerol and add 50ml of ddH 2 O to 100ml.
Sterilize 15 pounds of above-mentioned culture medium and glycerol solution with high temperature steam for 20min
2. Pour the plate: Heat the solid medium until it is fully melted. Before the temperature drops below 50°C and solidify, add Amp to 100ug/ml, shake and mix, and pour about 25ml into each plate. This process should be performed aseptically. Pour an LB plate without Amp.
3. After the medium is solidified, use an inoculation loop (after burning and sterilization) to inoculate each loop of the storage strains: E.coli InvaF’ and E.coli InvaF’ (pUC19) on the LB plate without Amp and containing On the LB plate of Amp, streak inoculation should prevent the medium from being scratched. The streaking method is shown in the figure:

After the first streak, the inoculation loop is burned, and then the second streak is carried out. The starting point of the second streak intersects with the first streak, and the third streak is carried out after the inoculation loop is burned. Swipe 5-6 times
4. After the inoculation is completed, put the petri dish upside down in an incubator at 37°C for overnight incubation. Using this streak method for inoculation can better ensure the formation of single colonies.
5. Compare the different responses of the two strains on the medium. Pick: A single colony of E.coli InvaF ‘E.coli InvaF’ (pUC19) was inoculated into 5ml LB liquid medium with and without Amp, respectively, and cultured overnight at 37°C with shaking.
6. Aspirate 0.5 ml of the bacterial solution under aseptic conditions, add 0.5 ml of 50% glycerol, put it in a sterile freeze-drying tube, mix well and store at -20°C.