What is hygromycin selection?
Hygromycin B is used for the selection of pro- and eukaryotic cells, stably transfected with the hygromycin resistance gene, as well as for the maintenance of the hygromycin phenotype of resistant cells.
How long does it take for hygromycin selection?
Cells transfected with a plasmid harboring the hygromycin resistance (hph) gene should be grown in complete growth medium for 48-72 hours post-transfection before selection antibiotic is applied.
Why do you heat shock the cells?
The heat shock step facilitates the entry of DNA into the bacterial cells. Recovery Broth is added to the cell suspension, and the bacteria are allowed to recover for 30 minutes at 37°C. This recovery period allows the bacteria to repair their cell walls and to express the antibiotic resistance gene.
Why are cells incubated at 42 C?
Plasmid DNA is added to half of the cells before they are “heat shocked” in a 42°C water bath. The heat shock step facilitates the entry of DNA into the bacterial cells. Recovery Broth is added to the cell suspension, and the bacteria are allowed to recover for 30 minutes at 37°C.
Why is electroporation better than heat shock?
Heat Shock Transformation. The advantages of using electroporation are the higher efficiency, more colonies, and much faster transformations compared to heat shock method.
Why do you incubate on ice for 10 minutes?
Keeping the cell temperature low will keep the temperature margin high, therefore increasing the effectiveness of “heat shock”. That is to increase the ‘competence’ of the bacterial cells, so that the plasmid can enter the cells more readily because of increased pore size resulted from the greater heat shock.
What is the difference between heat shock and electroporation?
Compatible cell types On the other hand, electroporation tends to be more efficient than heat shock. Hence, this method is amenable to a broader range of DNA amounts (from low to saturating concentrations), fragment sizes, and complexities.
What are the advantages of electroporation?
The main advantage of electroporation is its applicability for transient and stable transfection of all cell types. Furthermore, because electroporation is easy and rapid, it is able to transfect a large number of cells in a short time once optimum electroporation conditions are determined.
Why do you incubate at 37 degrees Celsius?
Mammalian cells operate optimally at 37 oC – molecular kinetics of enzymes and their substrate increase as the temperature increases, meaning a greater number of biochemical reactions can occur. Lower temperatures are less efficient.
Why are the cells incubated at 42 C?
Can I store plasmid at 4 degrees?
Plasmids provided as DNA, in suspension or on filter paper, should be stored at 4°C for up to two weeks.
What is hygromycin B?
Hygromycin B is an aminoglycoside antibiotic produced by Streptomyces hygroscopicus. It kills bacteria, fungi and higher eukaryotic cells by inhibiting protein synthesis.
What is the selection concentration of hygromycin B Gold in mammalian cells?
What is the selection concentration of Hygromycin B Gold in mammalian cells? The working concentration of Hygromycin B Gold in mammalian cell lines varies between 50 -200 μg/ml, with a few reported cases of up to 500 μg/ml.
What is the nucleotide sequence of the hygromycin B phosphotransferase from Streptomyces hygroscopicus?
Nucleotide sequence of the hygromycin B phosphotransferase from Streptomcyes hygroscopicus. M.C. The nucleotide sequence of a 1467 bp fragment of Streptomyces hygroscopicus DNA containing the gene (hyg) encoding a hygromycin B phosphotransferase (HPH) has been determined.
Does hygromycin B interfere with translocation?
It has been reported to interfere with translocation [1] and to cause mistranslation at the 70S ribosome [2] . Hygromycin B is used as a selective agent in molecular genetics experiments on a wide variety of eukaryotic and prokaryotic species [3-5] .