Experimental FAQ Collection (1)

Experimental FAQ Collection (1)

Can the PCR product obtained by BSP methylation amplification be directly sequenced, why do you need to construct TA clone and sequence it?

Since the degree of methylation of each CG site of genomic DNA is different, C which has not undergone methylation will be modified by bisulfite to U, and C will not change if methylation occurs, so if PCR products are produced Sequencing may result in a bimodal map at the original C site, and the degree of methylation cannot be determined. It is necessary to recover the PCR product, perform TA cloning, and randomly select 5-10 monoclonal sequencing methods to calculate each CG. The methylation status of the locus.

How to improve the transfection efficiency of mammalian cells?

For biotechnology research experiments, it is proposed to introduce foreign genes (or DNA sequences) into cells cultured in vitro (especially for various mammalian cells), allowing foreign genes to be expressed or even translated in cells for the intended purpose. In addition to ensuring high expression elements (promoters, enhancers, terminators, etc.) on plasmid vectors carrying genes, another important factor is to allow transfection efficiency of foreign genes to reach extremely high values, especially for genetic interference studies. In terms of transfection efficiency of more than 80%, it is a prerequisite for the successful completion of the interference assessment experiment.

On how to improve the efficiency of transfection, the following are some of the experience of the Ruisai team, the system is organized as follows

1. The importance of the state of the cell itself accounts for more than 70%. What aspects of cell growth status
1) Cell cycle: Cells in mitosis are often easier to ingest and express foreign DNA than quiescent cells. Therefore, the cells were plated on the day of transfection or the day before. It is also important that the cells should not be overgrown when transfected with the seed plate; in addition, for primary cells, mitogenic stimuli (such as viral transformation, growth factors, conditioned media, etc.) are also commonly used for activation. Therefore, it is necessary to observe how long the 293 series cells reach the dividing phase after the plating (12h?18h?24h?), but it cannot be overgrowth!
2) Cell fusion rate (number of densities): As long as there is space in the culture medium (dish), the cells will divide exponentially. For normal cells, the rate of cell growth is inhibited by contact with cell density (cancer cells are not restricted by this and continue to grow and superimpose each other). The depletion of nutrients and the accumulation of metabolic waste affect all cell growth, and cells are not suitable for transfection because of lack of nutrition. Personally think that the general 293 series cells are 50% dense, 16% can be transfected at 16-20h, and should not be transfected!
3) Passage: The number of passages refers to the frequency of batch passage of a cell line (usually within a laboratory). Certain cell lines are less stable than other cell lines and may change with prolonged culture time, depending on the cell line and culture conditions, so the same cell line with the same name is in physiology and morphology ( And the transfection ability) may also vary greatly in nature; in addition, the adherent cells are trypsinized by sub-plate subculture. This routine operation can cause serious damage to normal cell function, so it is necessary to pay attention to the plating of pre-transfection cells. Optimization of operation (such as the length of trypsin digestion, inactivation of trypsin, etc.). Textbooks say that cells are difficult to transfect within one or two generations after cryopreservation or until they are fully resuscitated.
4) Adherence and suspension: The difference between adherent cells and suspension cells is significant in transfection efficiency. Compared to adherent cells (such as HEK, CHO), suspension cells (such as HL 60, Jurkat) are very difficult to transfect, probably because of differences in intercellular membrane structure, but there is no explanation for a reasonable mechanism at the molecular level.
2, the quality of plasmid vector DNA is also an important factor in the quality of transfection
1) General principle: The quality of the purified vector is identified. When transferring, a known functional carrier must be used as a control.
2) Integrity of the vector: The vector element (promoter/enhancer/ORI, etc.) is verified in a written report.
3) Preparation quality of the carrier: Purity quality, whether there is cross-contamination of other carriers, residual contaminants (such as chloroform, alcohol, CsCl, endotoxin) in the preparation of the product may affect the transfection efficiency.
3. Culture conditions affect the success or failure of cell transfection experiments
1) DNA-transfection reagent complex: The pH value of the complex dilution in the transfection operation is very important (personal experience: in one batch of transfected cells, one well of the diluent is not the same tube as the other, the result is It is felt that the fluorescence of this hole is stronger than others. However, it is necessary to conclude that it is necessary to repeatedly verify the difference in cell status and random difference). Observe the color of the medium.
2) “Fresh” of the medium: ensure that the medium is fresh, avoid changing the medium system during the transfection experiment, and minimize the change of reagents or additives.
3) Nutritional guarantee: The serum and carbon dioxide concentrations will change slightly, which will have a great impact on the results.
4) Stop the transfection experiment when there is any possibility of contamination: contamination of chemicals, bacteria, fungi, and cross-contamination of cells and cells.

Using the same virus vector system for virus packaging experiments, why some people do very well, the second time is successful, some people often do not come out, the stability is very poor, is not low titer or no poison at all?

From our many years of experience in virus packaging of different carrier systems, we mainly have the following aspects.

First, several key nodes of viral packaging are cellular factors, vector systems (using mature commercial vector systems as much as possible), correctness of recombinant plasmid construction, plasmid extraction and purification, and packaging transfection control (24, 48 hours). The cell and fluorescence status judgment), the effect of the target gene on the viral packaging (gene size, sequence, protein functional toxicity, etc. will affect whether the packaging can be successful).
Second, if there is conventional experience in cell culture and cell transfection experiments, there should be no problem with cellular factors (cell culture personnel must pay attention to it. Before packaging or transfecting infection, we must pay attention to whether the cells are clean and fine, or whether the fullness is full. Good, uniform cell density is moderate), the vitality during the process of cell toxin production is an important part of the normal and out-of-virus virus titer (normally packaged virus, lentivirus, retrovirus packaging a cell) A virus granule, 1000 adenoviruses and 10,000 adeno-associated viruses), so the practice shows that the density of cells in the transfection of virus packaging should be controlled, so that the cell density is 90%-95 at the time of poisoning (72 hours). %about.
Thirdly, it is recommended to use a commercialized mature carrier system (not to use the system that is unclear or basically eliminated by few people). From the literature and our many years of practice, it can be concluded that such systems are stable and thus When analyzing the cause, try to spend less effort on the verification of the carrier system, which will make a lot of effort.
Fourth, as for the operational control details and transfection reagent factors in packaging transfection, as long as the relevant instructions and operating procedures are used during operation, there should be no major problems, so do not spend too much energy on this.
Fifth, another factor that needs to be focused on and investigated is the construction of the expression vector of the target gene and the extraction and purification. Does it result in the recombination of the plasmid vector or the deletion of a certain component, or contamination of other plasmids or debris? Is the extraction purified? Are you extracting other plasmids? To develop a habit of doing a negative control at the same time (whether the target gene vector and the negative control GFP vector are the same batch, it is recommended that the same batch, it is recommended to do this every time), if there is no problem in this link, there will be reorganization and deletion in the construction. Or the possibility of extraction and purification failure is not great.
Sixth, the implementation of the above factors, that is the last reason, the size of the target gene, the sequence of the situation, the functional toxicity of the protein of interest, etc. lead to the failure to package successfully (in practice, this situation is there, we occasionally encounter The possible reason is that some gene expression is toxic to the packaging cells after translation, resulting in abnormal cell state. Then what we can do is to change the virus packaging carrier system and try to package other carrier systems. However, the chances of this happening are very low. If you do 100 genes, you may not be able to touch one.
Therefore, in general, we should pay attention to the packaging materials - cells and expression plasmids when carrying out virus packaging experiments (to verify the cells and vector systems taken over, often there are problems with packaging cells, empty expression vector plasmids, we have to periodically Purification and production of packaging cells, empty expression vector plasmids, cell culture, let it "white fat, full of vitality", the expression vector of the target gene is well purified, the ratio between the plasmids is appropriate, the virus packaging experiment can still have better results.

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