Introduction
Plasmids, circular pieces of DNA that can replicate independently from the host cell’s DNA, have been extensively used to transform cells in biological research to study the effects of particular genes of interest (Lau et al., 2013). A plasmid’s basic components include a replication origin, a DNA marker, and a multiple cloning site, which are sufficient for the plasmid to replicate itself and help the transformed cell to exhibit characteristics indicating successful transformation, such as drug resistance (Molecular Biology Laboratory Manual). In order for the plasmid vector to enter the bacterial cell, the cell must be competent. A common method of increasing cell competency is heat shock, which increases the permeability of …show more content…
Cells such as DH5 α E Escherichia coli that have inactive N-terminals of β-galactosidase can combine with inactive C-terminal β-galactosidase from empty plasmid vectors such as pGEM to create a complete active β-galactosidase, which converts the clear chromogenic substrate X-gal into a blue substance (Mogalisetti and Walt, 2015). However, inserted genes in plasmid vectors disrupt α –complementation from occurring, preventing X-gal from changing color. Thus α –complementation easily distinguishes bacteria with the gene of interest from those without …show more content…
300 μL ice cold mixture of 50 nM glucose, 25 mM Tris-Cl, 10 mM EDTA, and RNase A was put into each tube to cleave the plasmid RNA and provide nutrients for the plasmids’ survival. Then, the bacterial cell walls were broken with 300 μL of a 0.2 NaOH and 1% SDS mixture and 300 μL of 5% potassium acetate and the tubes were stored on ice for 5 minutes. After centrifuging, 600 μL of isopropyl alcohol were added the supernatants of each tube. After cleaning the pellets with 70% ethanol, 45 μL nano pure water was used to dissolve each pellet. Different 30 μL mixtures of pGEM, pGEM-luc, Master Mix N and Master Mix H were incubated at 37°C overnight and kept at 4°C for the following 6
Plasmids, circular pieces of DNA that can replicate independently from the host cell’s DNA, have been extensively used to transform cells in biological research to study the effects of particular genes of interest (Lau et al., 2013). A plasmid’s basic components include a replication origin, a DNA marker, and a multiple cloning site, which are sufficient for the plasmid to replicate itself and help the transformed cell to exhibit characteristics indicating successful transformation, such as drug resistance (Molecular Biology Laboratory Manual). In order for the plasmid vector to enter the bacterial cell, the cell must be competent. A common method of increasing cell competency is heat shock, which increases the permeability of …show more content…
Cells such as DH5 α E Escherichia coli that have inactive N-terminals of β-galactosidase can combine with inactive C-terminal β-galactosidase from empty plasmid vectors such as pGEM to create a complete active β-galactosidase, which converts the clear chromogenic substrate X-gal into a blue substance (Mogalisetti and Walt, 2015). However, inserted genes in plasmid vectors disrupt α –complementation from occurring, preventing X-gal from changing color. Thus α –complementation easily distinguishes bacteria with the gene of interest from those without …show more content…
300 μL ice cold mixture of 50 nM glucose, 25 mM Tris-Cl, 10 mM EDTA, and RNase A was put into each tube to cleave the plasmid RNA and provide nutrients for the plasmids’ survival. Then, the bacterial cell walls were broken with 300 μL of a 0.2 NaOH and 1% SDS mixture and 300 μL of 5% potassium acetate and the tubes were stored on ice for 5 minutes. After centrifuging, 600 μL of isopropyl alcohol were added the supernatants of each tube. After cleaning the pellets with 70% ethanol, 45 μL nano pure water was used to dissolve each pellet. Different 30 μL mixtures of pGEM, pGEM-luc, Master Mix N and Master Mix H were incubated at 37°C overnight and kept at 4°C for the following 6