Cell lines verification is of the utmost importance. Skipping the verification step can lead to invalid research data, leading to lost time and money and retractions of publication. Herein, we have collected the Frequently Asked Questions on cell lines verification, hoping to provide research tips on cell line verification.
Low transfection efficiency and no positive cells screened in flow sorting?
Mycoplasma contamination can reduce the transfection efficiency of cells. In addition, transfection reagents and cell species are the main factors affecting cell transfection efficiency.
Why do the cells that are placed on 96-well plates grow slowly, and some cells even die?
Before starting to study the function of the protein, it is best to find out relevant references to determine whether the knockout of the gene encoding the target protein will affect the growth and proliferation of the cell. Some gene knockouts can inhibit the growth of cells, and some have lethal effects on cells, making experiments difficult. Therefore, it is necessary to consult the relevant literature on your gene and model species.
Sometimes the target band cannot be amplified by PCR when doing genotyping, why?
Before knocking out, it is best to perform short tandem repeat (STR) analysis identification on cells (especially cells from outside sources) and make sure that the cells used in the experiment match those intended for use according to the research strategy.
After eliminating any cell source problems, you can optimize PCR conditions by setting temperature gradients, adding DMSO, switching to high-fidelity enzymes, and replacing primers.
Why do some cells show mutations at the DNA level after gene knockout, but the protein level (Western blot) does not change significantly?
Only when a frameshift mutation occurs in the genome, the encoded amino acid after the mutation site will change widely, which in turn causes the loss of the normal structure and function of the protein. Therefore, several knockout cell lines can be screened for by Western blot (WB) detection. The amino acid recognition sites of antibodies may be different for various clone numbers, and the antibody instructions should be read in detail before Western blot detection to pinpoint the amino acid sites they recognize and to avoid false-negative results. Western blot can also be used to validate the knockdown effect in conjunction with later protein function experiments.
Cyagen Custom Cell Line Generation Services:
With decades of experience and thousands of successful projects, Cyagen is a strong partner in cell model design and preclinical research. Cyagen can provide many custom cell lines, such as:
>> Knockout Cell Lines
>> Knockin Cell Lines
>> Point Mutation Cell Lines
>> Overexpression Cell Lines
Upon request, we can deliver homozygous, heterozygous, or negative clones. We can additionally provide professional experiment reports and quality inspection reports.
