Question 10, Experimental Design Question. Invite students to read a piece of material.

Nucleic acid restriction enzymes are one of the most important tool enzymes in recombinant DNA technology, which is a class of nucleic acid hydrolases that can recognize specific base sequences in double-stranded DNA and endolyze DNA strands. The most suitable restriction enzymes for genetic engineering operations are the second type of endonucleases, which are characterized by the fact that the breaking points of the cleaves are all on the inverted repeats, also known as palindromic sequences, such as:

EcoR I

5’-G↓AATT C-3’

3’-C TTAA↑G-5’

BamH I

......

Please design a restriction enzyme digestion identification experiment for plasmid DNA.

"Fortunately, I have done other experiments on nucleic acids, the experimental design of this nucleic acid restriction enzyme, let's make it up!"

Lan Jie has not done restriction enzyme experiments with his own hands, and he wants to do it very much, but restriction enzymes are expensive biochemical reagents, and the coach does not give them to him.

At this time, Lanjie can only rely on the operation experience of similar experiments and rich theoretical knowledge reserves to make reasonable arrangements.

The first step is the digestion reaction.

The second step is the preparation of the electrophoresis gel plate.

The third step is electrophoresis and sample addition.

The fourth step is electrophoresis and identification.

Langer first devised four main experimental procedures.

Materials required include: plasmid DNA, reaction buffer, BSA, restriction enzymes, ......

The experimental equipment required includes: electrophoresis tank and electrophoresis instrument, ultraviolet detector, electric heating constant temperature water bath......

Then it's a matter of refining each step and writing out the details of the experiment.

Once the design is complete, it should be theoretically feasible. ’

Lan Jie checked the test paper from beginning to end, and just after the check, the bell rang and the paper was handed in.

Tomorrow will be the experimental test, Lei Tiangang said to his teammates: "Students who feel good about themselves in the theory test can communicate with our coaching team about the theory test." For those of you who feel average about yourself, go back to the dormitory to have a good rest, adjust your state, and actively prepare for tomorrow's lab exams. ”

Lei Tiangang said, half of the players went back to rest, and the remaining half of the players answered with the coaches.

"Yes, yes, that's what I wrote!"

"Haha! I'm getting it right again!"

"Huh?

"Wrong again?"

"After the sixth question, am I all wrong?"

"I'm so mentally broken!"

......

There is a certain risk in answering the questions before the contest is over.

Students who feel good about themselves find that the accuracy rate is higher after completing the answers, and of course they will feel better about themselves, and their confidence and morale will be doubled, which will be conducive to tomorrow's experimental competition.

Otherwise, it will explode.

"So, my theoretical score will not be lower than 95 points?" After answering the theory questions, Lan Jie found that his feeling was quite accurate, and if he could get a score of no less than 95% of the theoretical score, it would be very impressive.

"And my theoretical score will not be higher than 50 points, I hit the street, I don't want to participate in the experimental competition......" a certain team member sighed in a sophomore high school, and his mood was extremely low.

The students who can be selected for the sheep middle school competition team are all top students in the field of biology, under normal circumstances, these top students have handed in the papers, and before they have the right answers, the valuation of their scores is basically eight or nine.

After the answer, the actual value is 80%~90% of the expected value, which is very common, and the accuracy of most students' evaluation is within this range.

I scored 90 points and scored 30 points. Or estimate 30 points and score 90 points. Both extremes are rare.

The sophomore student of the sheep middle school competition team finished the theory test, and he felt extremely good, he felt that he could score 90 points, and after completing the answers, he found that his actual value would not exceed 50 points.

The sophomore in high school is still too young, and it is good for young people to be passionate and optimistic, but if they are carried away by excessive passion or blindly optimistic, they will hurt themselves.

Lan Jie comforted: "Senior brother, you were selected for our sheep middle school competition team when you were a sophomore in high school, and it is already great to come to the national finals." You still have a chance next year, and I'm sure you'll be stronger next year. So you have to cheer up, our students in the sheep don't lose points, you have to participate in tomorrow's experimental competition, it's a valuable experience. If you come back to the national finals next year, you will be a veteran with actual combat experience. ”

"Senior Lan Jie, you're right, I just said that I was angry, I will definitely participate in tomorrow's experimental competition. On the one hand, it is to accumulate experience in the national finals, and on the other hand, if the other players play abnormally in the experimental match, and I play at a high level, then I will have a chance to turn the tables!" "Young people are young people after all, and it is only a matter of seconds to go from optimism to pessimism, and then from pessimism to optimism. The optimistic sophomore did not give up hope, and he pinned his hopes on other players to make mistakes and give them a chance.

Lan Jie was happy: "Haha, junior, you can think so, but it's too right! You just wait, wait for the other players to give you a chance." I'm sure someone will give me a chance. ”

The subject competition is an individual show, and only individual awards are awarded by the organizer.

A certain province or a certain middle school has won several awards, and whose competition strength is the strongest is an unofficial folk statistics.

Each year, sixty or seventy national awards are awarded in proportion to the national competition, and the average is two national awards in each province.

There are so many middle schools and so many students in each province, so it is very difficult to get the National One Award.

Competitors are not only students from other schools or provinces, let's say Lan Jie's Yang Zhong student competition team, Lan Jie and other Yang Zhong students are teammates, but also competitors.

The national finalists do not count the team's total, which means that teammates are of little value.

As competitors, teammates are more valuable.

To be reasonable, no one wants to go against the wind. But because of the poor performance in the first half, I had to fall into a headwind, which also happens often.

The players against the wind all hope to turn the tables against the wind. The sophomore is this kind of person, and there are many players who are similar to the sophomore.

Lan Jie, who is temporarily in the tailwind game, of course he will not give a chance to turn the headwind around.

The next day, the experimental match.

The full score of the experimental competition is 100 points, which is the same as that of the theory competition.

Energetic, Lan Jie came to the designated laboratory to check his personal experimental equipment and experimental reagents.

Centrifuge, electrophoresis tank, gel plate (agarose gel is prepared by the organizer in advance), staining solution, latex gloves, 4 pipette guns, gun box, relevant reagents for plasmid extraction, topoisomerase (0.5U/ul), 10*loading buffer, Marker DNA, enzyme working solution, distilled water...... These are Langeres' personal laboratory equipment and experimental reagents.

Utilities are miniature ultraviolet spectrophotometers, water baths, and ultraviolet imagers.

The contestants were required to extract the plasmids of the given bacteria, design experiments, verify the role of topoisomerase I, write a brief experimental design idea, and clearly explain the ratio of solutions in each system......

There are certain similarities between this practical experiment in molecular biology and the last experimental design question in yesterday's theory competition! There is a correlation between the theory competition and the experimental competition, and the level of the teachers who came up with the questions in the national finals is high. Lan Jie immediately began to design experiments, he had not done exactly the same experiment, but in the biological laboratory in sheep, he had done experiments with the same principle and a high degree of specific operation.