When doing the transfection experiment, Xiaobian holds a belief that it is necessary to give the cells a "color" look (fluorescence), but do you know? In this simple experimental process, small cells have great energy. During the experiment, the cationic liposome acts as a “porter†to “grab†the plasmid from the extracellular to the cell, and the cell is a plasmid. The copying, transcription, and translation provide "places and raw materials", and they also follow the "lighting and coloring", but there are some problems in the process of "coloring". The following two aspects are summarized: âž™ poor cell status âž™ Low transfection efficiency In the principle of "the technician who will not solve the experimental problem is not a good technician", the following small series will share their own experiences. A: Analysis: Mainly the reason for the preparation of the plating and reaction solution ⇒ Cell state is the key to the whole experiment. If the cell state is not good, the cell state after transfection is of course not good. 铺 tiling: too much or uneven, the cell is too long to be in a bad state è„‚è´¨ Liposome itself is toxic. If the amount of liposome added is too large, it will lead to poor cell status or cell death. It is recommended to do a preliminary experiment before the formal experiment to explore the distribution of plasmid and liposome. ratio. A: Analysis: mainly the cause of the cell and the problem in the "handling" process ⶠ. Cell state is still the most important thing. If the transfection effect is not ideal for a period of time, you must change a batch of cells; â·. After adding the target plasmid mix gently, too hard it will destroy the structure of the mixture, ⸠. The ratio of plasmid to transfection reagent: too high a part of the plasmid can not enter the cell, too low "porters" too much, toxic and wasteful; . â¹ suction, liquid level in the pipette tip note playing a liquid, is completely played; . ⺠After the reaction solution with a good wait 20min, the plasmid will only be fully wrapped; â» The reaction solution was added dropwise added to the cells, so that the "liposomes + plasmid" uniform distribution; â¼ . Repeated freezing and thawing of the plasmid affects the concentration, and minimizes the number of freeze-thaw cycles; the liposome is stored at -20 ° C, and should be replaced immediately after use; â½ need to detect it if necessary concentration of plasmid is accurate.; It takes 48 hours to transfect, can you know the effect of transfection in advance? According to experience, the fluorescence effect at 24h after transfection reaches at least the level of the figure below, so basically don't worry~  Have you learned to "color" cells? Welcome to ask more experimental questions, everyone to discuss! Long press and pay attention John Dory Fish,Frozen Fish Fillets,John Dory Fish Fillet,Cream Dory Fish Fillet Skin Zhejiang Zhoushan Jiaze Aquatic Co., Ltd. , https://www.tianjia-aquatics.com
The secret of the experiment - how to do the brightest cell transfection?