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    Home > Biochemistry News > Biotechnology News > Recombine the construction of prosurds.

    Recombine the construction of prosurds.

    • Last Update: 2020-10-22
    • Source: Internet
    • Author: User
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    recombined prosurfic construction is commonly usedmolecular biology means, in fact, is only the most basic method, generally a week at the same time to build three or two groups of prosultons is no problem.


    in advanced experiments in China, is also mostly done by experimenters. But there are some basic skills to master. Here will share my heart in everyone, this is my own experience in the first line of work over the past few years, in order to provide reference for Guyou, so that we can take fewer detours in the experiment.


    relates to the following:


    left;">1) clone" theof the genease cut-point problem


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    I now make one-by-one identification of the selected clone bits before each build, such as selecting NdeI and HindIII as clone bits, and then monoenzyme-cutting the enzyme cut points of the two enzymes on the protons. Single-enzyme cutting identification can be effectively cut, and then issued a quotation synthesis order, for the synthesis of the lead;



    is generally double-enzymatic in a universal buffer, but these two enzymes are not as efficient at enzymatic cutting in a universal buffer, which can lead to the existence of a single notch of the prosaic fragments, so that in the connection reaction, even under the presence of the external DNA fragments, the single-gap proton fragments can be more quickly and efficiently connected.

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    experimental case study 2: I used XhoI and HindIII enzyme incussion points to build recombinant plasmogenes, after double-enzyme cutting of plasmed particles, directly connected, not the above two-step identification, each result full of plaque. But there is no positive. Later, after the protons were identified, the XhoI enzyme cut point was found to be damaged. Another month without progress, a waste of energy and medicine.


    the lesson of blood. Because at that time did not notice: single-cut granules are a band, double-cut granules are also a band, electrophoresis behavior is the same, can not be distinguished. If you do any of the above identification will know that the problem is there, ha ha.


    case study 3: A large doctor in this laboratory known as rigorous experiment, with KpnI and HindIII to build recombinant plasmrus, a month fruitless, only negative spots, not positive spots, after suspected KpnI enzyme failure. Anger KpnI and throw away all KpnI enzymes in the lab without my knowledge. After I learned, asked him to do the above two identification experiments, he quibbled that no, after the identification of Hind III bit failure.


    finally he scolded me for keeping a hand secretly,not conferring. Oh, he doesn't blame himself for not thinking, just wood head to do experiments, but bite a mouthful of bell-ringing people, and I didn't know what problems he encountered before that. Oh, do you think it's wrong? This world! It can also be seen that the exchange between laboratory personnel is very important.


    after writing the first two questions two weeks ago, finally was able to take the time to write the third question, which is relatively simple after doing the above two aspects of the work.


    , when connected The concentration ratio of the two fragments is the problem


    general experimental instruction manual says that the protons: fragments are 1:3 (molar ratio), in practice I thought 1:5 or even 1:10 is appropriate. Do a good job of "one, two", 16 degrees C after 10 hours, each time can be effectively connected. Of course, there is the E. coli sensory state problem, we used to do it ourselves, now lazy to do, all with the "day for the times company" products, feel good (especially declare that I am not the company's inside line, ha ha).


    here describes a method for estimating DNA concentration at the location: DNA can be detected by ultraviolet method, or electrophoresis can be compared to marker estimation, in the case of less precise requirements, you may wish to try the following method:


    1. Take a flat dish.


    2. Thinly pour a thin layer of < a href"" containing EB> agargum, solidified (4 degrees C can be stored for a week).


    3. The back of the flat dish can be drawn as a small square.


    4. A small grid of mid-point 1 ul sample.


    5. Another small grid point 1 ul DNA "standard (I usually use Takara 2000 DNA lander, 1 ul 60 ng)


    6. After cool drying, the UV lamp can be estimated according to the brightness.


    OK, I can estimate the concentration so much when I connect, you can know the concentration of two fragments in 5 minutes. In fact, the connection fragment concentration ratio can be filled in a range, 1:5 to 1:10 can be, so the above estimation method is available in this case.


    (Responsible Editor: King Kunlun of The Great Han)

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