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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. < p style is "text-align: left;" > 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. 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. , when connected The concentration ratio of the two fragments is the problem 6. After cool drying, the UV lamp can be estimated according to the brightness.
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