DNA sequencing
DNA Sequencing
As of last few weeks, the transformation lab is performed to convey and purify a given protein. However after further research scientists found out that Transformation is not only used to purify protein but also to find out contents that are stored in a given plasmid. The objective of the lab that is to be performed involves a procedure that determines the identity of an unknown gene replicated in a plasmid. To begin this procedure two to four colonies of bacteria is added to two micro tubes filled with CaCl2. In this process the UV light was used periodically to observe the condition of the bacteria in the micro tubes. After a few times of incubation on ice, hot water and room temperature, LB nutrient broth was added to the mixture to help the bacteria with its growth. These mixtures were then added to the appropriate agar plates for observations and place them in the incubator. After 10 – 16 hours of incubation, to obtain conclusive results, the DNA sequence must be read. In order to achieve this task, the bacterial cells will break open and isolate the DNA in it. This process is done by using several procedures such as Miniprep, Quantification, and sequencing. After performing these procedures, it will clearly show the contents of the unknown plasmid. The expected end results of this lab will be the sequencing of the pRSETA plasmid and it contains ampeciline resistance gene, a constitutive T7 promoter that regulates expression of the gene of interest, a multiple cloning site that contains many enzyme sites that required cutting and pasting DNA sequences.
As of last few weeks, the transformation lab is performed to convey and purify a given protein. However after further research scientists found out that Transformation is not only used to purify protein but also to find out contents that are stored in a given plasmid. The objective of the lab that is to be performed involves a procedure that determines the identity of an unknown gene replicated in a plasmid. To begin this procedure two to four colonies of bacteria is added to two micro tubes filled with CaCl2. In this process the UV light was used periodically to observe the condition of the bacteria in the micro tubes. After a few times of incubation on ice, hot water and room temperature, LB nutrient broth was added to the mixture to help the bacteria with its growth. These mixtures were then added to the appropriate agar plates for observations and place them in the incubator. After 10 – 16 hours of incubation, to obtain conclusive results, the DNA sequence must be read. In order to achieve this task, the bacterial cells will break open and isolate the DNA in it. This process is done by using several procedures such as Miniprep, Quantification, and sequencing. After performing these procedures, it will clearly show the contents of the unknown plasmid. The expected end results of this lab will be the sequencing of the pRSETA plasmid and it contains ampeciline resistance gene, a constitutive T7 promoter that regulates expression of the gene of interest, a multiple cloning site that contains many enzyme sites that required cutting and pasting DNA sequences.