Preparation of electroporation-transformed receptor E.coli TG1 cells
This experiment was mainly used to prepare receptorized E.coli TG1 cells.
Operation method
Preparation of electroporation-transformed receptor E.coli TG1 cells
Principle
The main principle is to make the cell more permeable by treatment, which, intuitively speaking, makes some holes on the surface of the cell membrane to facilitate the entry of exogenous genes or vectors into the receptor cell. Due to the fluidity of the cell membrane, such holes will be repaired by the cell itself.
Materials and Instruments
TG1 cells Move I. Reagent Preparation SB medium Tryptone for bacterial culture 20g Yeast extract for bacterial culture 5g NaCl 0.5g Deionized water 950ml 250mmol/L KCl solution 10ml Steps 1. Select a TG1 clone from fresh LB plate culture and inoculate it in 10ml SB medium. 2. Shake the bed at 37℃ for overnight incubation. 3. Take 2.5ml of culture and transfer it into 0.5L SB, add another 10ml of 20% glucose and 5ml of 1mol/L MgCl2. 4. Incubate at 37°C until OD600=0.7-0.8. 5. The culture was placed in an ice bath for 15 min, then centrifuged at 4°C for 20 min at 4000 r/min, and the supernatant was discarded. 6. Resuspend the bacteria in 1/2 volume of pre-cooled 10% glycerol, centrifuge at 4000r/min for 20min at 4℃, discard the supernatant. 7. Repeat step (6). 8. Resuspend the bacteria in 15 ml of 10% glycerol and transfer to a 50 ml centrifuge tube. centrifuge at 3500 r/min for 15 min at 4°C. Carefully remove the supernatant to obtain 4 to 5 ml of bacteria. Blow and resuspend rapidly; 9. Dispense into small portions of 200 μl or 40 μl. The operation should be carried out in an ethanol/dry ice bath. Store at -70°C. 10. test the transformation efficiency of the prepared sensory bacteria with the pUC19 plasmid, which should reach 109 cfu/μg DNA. Caveat 1. It is crucial to select low-generation strains, and never save the activated strains after each activation, not to mention keeping the strains at -20 degrees for a long time. 2. E. coli can be stored in the refrigerator for about one month after overnight growth on the plate; inoculation of fresh colonies or bacterial fluids is conducive to synchronous and rapid growth of bacterial cells, so that the bacterial population is in the sensory state after reaching a certain OD value (0.375) for most of the cells. 3. The growth of E. coli requires oxygen, and the purpose of vigorous oscillation is to increase the dissolved oxygen of the medium to facilitate bacterial growth. 4. It takes about 2.0~2.5 hours to reach the early and middle stage of bacterial logarithmic growth, and this step is critical, if it exceeds this stage, the transformation efficiency will decrease dramatically. 5. The purpose of low temperature operation is to make the cells no longer grow and maintain their sensory state. 6. Cells are fragile when being washed, so be gentle when suspending and settling, and use a pipette gun to gently pipette. 7. Receptor cells stored in the refrigerator at -80°C have a rapidly decreasing transformation rate after 6-8 weeks. A closed-loop plasmid such as pUC18/19 with a known standard and concentration can be used to identify the transforming ability of the sensory cells. Common Problems Source Handbook of Laboratory Techniques for Industrial Microbiology For more product details, please visit Aladdin Scientific website.
NaCl Tryptone Yeast extract Deionized water KCl NaOH Glucose solution MgCl2 Glycerol
Thermostatic rotary shaker Triangular flasks Centrifuge tubes Low-temperature high-speed centrifuge