Team:LN-Shiyan-China/Experiments
First of all, we use the PCR to proliferate the plasmid provided by IGEM. PCR is a kind of mechanism that proliferate the target DNA segment beyond the capacity of the nature, the velocity of the proliferation of the PCR mechanism can be up to 100,000 generations per day, thus provide us with plenty of plasmid for the experiment. Then the module is used with the purified water, 10*buffer, dNTP, F and R primer to synthesize the target opdA enzyme’s DNA. Then the result solution was put into the PCR again, waiting for later purification by using the reagent----Gel extraction kit. When waiting for the PCR with the solution we made earlier, the medium for the coli was made by mixing the soybean powder with water, the result 1 liter of solution is then equally divided into four parts because of the limitation of the container which has the maximum capacity of 250ml. Two of them are simply made by mixing water and soybean powder, while the rest two were added agar thus created two solid medium. Because of the rigid requirement of the coli and avoid contamination, the for containers with the medium are put into an autoclave to eliminate the potential contamination in the container. The next step is preparing for the separation gel by mixing the agarose and water, result in the solution with the volume of 30ml. The solution is then put into the oven to mix the agarose and the water entirely. After taking it out of the oven and cooling the solution, the dye which make the DNA appear when exciting light was lit upon the gel is put into the solution. The solution is put into the model with a comb, in order for the gel to concrete. With the previously made separation gel, the DNA model is put in to the hole left by the comb and the model is put into another hole. The electrophoresis is then used to extract the target DNA segment in the solution. The resulting gel has many stripes and one of them contains the target DNA segment. In order to purify the DNA segment, the stripe need to be neatly cut out of the gel. Then the Gel extraction kit is used to purify the DNA by following the instructions in the Gel extraction kit.
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