Question 10, experimental design questions. Please read a piece of material.

Nucleic acid restriction enzymes are one of the most important tool enzymes in recombinant DNA technology. They are a class of nucleic acid hydrolase that can recognize specific base sequences in double-stranded DNA and hydrolyze the DNA chain in an endogenous manner. The most suitable restriction enzyme for genetic engineering is the second type of endogenous enzyme. An important feature of this type of enzyme is that the cleavage break points are all on the reverse repeat sequence, 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 enzymatic identification experiment for plasmid DNA.

‘Fortunately, I have done other nucleic acid experiments, and the experimental design of this nucleic acid restriction endonuclease is compiled!’

Ranger has not done any restriction enzyme experiments with his own hands, and he also wants to do it. However, the restriction enzyme is an expensive biochemical reagent and the coach does not give it to him.

At this time, Lanjie could only rely on his operational experience and rich theoretical knowledge reserves to make reasonable arrangements.

The first step is enzyme cleavage reaction.

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

The third step is electrophoresis to add samples.

Step 4: electrophoresis and identification.

Ranger first designed four main experimental steps.

The required experimental materials include: plasmid DNA, reaction buffer, bsa, restriction endonuclease...

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

Then, refine each step and write down specific experimental operation details.

‘It should be feasible in theory after designing it.’

Langjie checked the test paper from beginning to end. As soon as the inspection was finished, the ringtone rang and handed over the paper.

The experimental exam will be held tomorrow. Lei Tiangang told his team members: "Students who feel good about themselves in the theory exam can communicate with our coaching team about the theoretical exam. Students who feel average in themselves, you can go back to the dormitory to have a good rest, adjust their status, and actively prepare for tomorrow's experimental exam."

After Lei Tian said that half of the players went back to rest, and the remaining half of the players met the coaches' answers.

"Yes, yes! That's how I wrote it!"

"Haha! I'll do the right question again!"

"Ah? Wrong?"

"Wrong again?"

"After question six, am I wrong?"

"My mentality is broken!"

...

There are certain risks when the competition is not yet over.

After completing the answer, students who feel good about themselves find that the accuracy rate is higher, so their self-feeling will of course be better, and their confidence and morale will double, which will be beneficial to tomorrow's experimental competition.

Otherwise it will explode.

"It seems that my theoretical score will not be lower than 95 points?" After answering the theoretical question, Ranger found that his feeling was quite accurate. If he could get a score of no less than 95% of the theoretical score, it would be very considerable.

"And my theoretical score will not be higher than 50 points. I missed. I don't want to participate in the experimental competition anymore..." A player in his second year of high school sighed and felt extremely depressed.

The students who can be selected for the Sheep Middle School Competition are all top students in the biological field. Under normal circumstances, these top students hand in the paper, and before answering the answer, their valuation of their scores is basically the same.

For the answer, the actual value is 80% to 90% of the expected value, which is very common. Most students' estimation accuracy is within this range.

Estimated 90 points, get 30 points, or get 30 points, get 90 points. Both extreme situations are rare.

This sophomore high school student from the Sheep Middle School Competition team finished the theoretical questions and felt extremely good. He felt that he could get 90 points. After finishing the answer, he found that his actual value was very likely not to exceed 50 points.

The junior in the second year of high school is still too young. Young people are passionate and it is a good thing to have an optimistic attitude, but being blinded by excessive passion or blindly optimistic will hurt yourself.

Lanjie comforted, "Senior brother, you were selected for our Sheep Middle School Competition Team when you were in your second year of high school. It is already amazing to come to the national finals. You still have a chance next year, and I firmly believe that you will become stronger next year. So you must cheer up. The students in our Sheep will not lose points. You must participate in tomorrow's experimental competition. This is a valuable experience. If you come to the national finals again next year, you will be a veteran with practical experience."

"Senior Langjie, you are right. I just said something angry. I will definitely participate in tomorrow's experimental competition. On the one hand, it is to accumulate experience in the national finals. On the other hand, if other players perform poorly in the experimental competition and I perform at a higher level, then I will have a chance to turn the tables!" After all, young people are young people. From optimism to pessimism, and then from pessimism to optimism, it only takes a few seconds. The second-year junior who was optimistic again did not give up hope. He hoped that other players would make mistakes and give opportunities.

Langjie was happy: "Haha, junior brother, you can think so, but it's so correct! Just wait, wait for other players to give them a chance. I believe that someone will definitely give them a chance."

The subject competition is a personal show, and the organizer only awards.

A certain province or a certain middle school won several awards. Which one has the strongest competition strength? It is an unofficial folk statistics.

Every year, the national election awards of sixty or seventy countries will be awarded in proportion. On average, in each province, there will be only two countries and one award.

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

The competitors are not only students from other schools or provinces. Let’s say that Langjie and their Sheep Middle School competition team. Langjie and other students from Sheep are teammates and are also competitors.

The national finals do not count the total score of the team, which means that teammates are of no value.

As a competitor, teammates are more valuable.

To be honest, no one is willing to go against the wind. But because of his poor performance in the first half, he had to fall into a headwind, which often happens.

All the players who are against the wind want to turn the tables against the wind. The second-year junior is like this, and there are many players who are similar to the second-year junior.

Ranjie, who is temporarily in a downwind game, certainly will not give him a chance to make the side turn around.

The next day, the experimental competition.

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

The energetic Ranger came to the designated laboratory to check his personal experimental equipment and experimental reagents.

Centrifuge, electrophoresis tank, rubber making board (the agarose gel was prepared in advance by the organizer), staining solution, latex gloves, 4 pipettes, gun box, relevant reagents for plasmid extraction, topoisomerase (0.5u/ul), 10*loading     buffer, marker     dna, enzyme working fluid, distilled water... These are Ranger’s personal experimental equipment and experimental reagents.

Public equipment is a miniature ultraviolet spectrophotometer, water bath pot, and ultraviolet imager.

The experiment required the contestants 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 solution ratio in each system...

This practical experiment in molecular biology has certain similarities with the last experimental design question in yesterday's theoretical competition! The theoretical competition is related to the experimental competition, and the national finals' question-setting teacher is of high level. Langjie immediately started designing the experiment. He had never done exactly the same experiment, but in the biology laboratory of Sheep, he had experiments with the same principles and similar specific operations.
Chapter completed!