The principle of recombinant DNA transformation experiment: After the recombinant molecule with foreign DNA is constructed in vitro, it needs to be introduced into an appropriate host cell for propagation or to express a protein with a certain biological activity. Receptors that can be transformed into recombinants include animal cells, plant cells, and microbial cells. There are many different ways to introduce into recipient cells: transformation (transfection), microinjection and electroporation. Different introduction methods can be selected according to different receptors. In general, lower eukaryotic cells such as prokaryotes such as bacteria and yeast can be introduced by transformation, while higher animal and plant cells require microinjection or electroporation. After the bacterial surface is treated with CaCl2, the cell wall is partially lost or dissolved, and DNA molecules can enter the cell through the plasma membrane. The way it enters the cell is that the complete double-stranded DNA molecule is first adsorbed on the cell surface, the double-stranded DNA molecule melts into a single strand, and the single-stranded DNA molecule enters the recipient cell and the other is degraded. The single-stranded DNA that enters the cell is copied into double-stranded circular DNA, which is replicated simultaneously with the replicon and is transcribed and translated. Experimental method (using CaCl2 to prepare fresh competent cell transformation method): Competent bacteria preparation (1) Pick the activated colonies on the plate and inoculate them in 2 ml of LB culture medium, and incubate at 370C overnight. (2) Pick up the overnight culture bacteria at 1% and inoculate them in 50ml LB culture medium, shake and culture at 370C for 2 ~ 2 half-hours (OD600 is about 0.2 ~ 0.4). (3) The culture was cooled in an ice bath for 10 minutes. (4) In a centrifuge tube, centrifuge at 40C5000rpm for 5min, collect the bacteria, and pour the culture solution as clean as possible. (5) Suspend the bacteria in 10 ml of 100mmol / L CaCl2 solution, place in an ice bath for 20min, centrifuge at 40C 5000rpm for 5min, and collect the bacteria. (6) Suspend the cells in 1-2ml of 100mmol / L CaCl2 solution, store at 40C, and transform into competent cells after 12-24 hours. Conversion experiment (7) Take 200ml of competent bacteria, add the recombinant DNA to be transformed (volume should be less than 10ml, DNA <50ng), mix well and place in an ice bath for 30min (generally a positive control and a negative control should be done at the same time. The positive control is added Known amount of competent bacteria without recombinant plasmid DNA is used to detect transformation efficiency. Negative control is competent bacteria without plasmid DNA at all, used to detect the presence or absence of contamination of competent bacteria and to detect antibiotic activity). (8) Place in an ice bath for 30 min, transfer to a 420C water bath for 90 s, re-ice bath for 1-2 min, add 800 ml of culture medium, and shake at 370C for 45 min. (9) Spread an appropriate volume of transformed competent cells evenly on the dish. (10) After the liquid in the dish has been absorbed, turn the dish upside down and incubate at 370C for 12-16 hours to produce colonies. More DNA transformation experiment methods are in Beijing Jiehui Biology, please call for consultation.
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