Wk5 Transformation BackgroundSP15
Transformation of E. coli with your unknown plasmid
Learning Goals: Insert your uncut unknown plasmid into chemically competent DH-5 E.coli cells and use antibiotic resistance to confirm the success of the transformation. You should familiarize yourself with the various methods of transformation and the advantages/disadvantages of each type. You should also understand how heat shock transformation works and how chemically competent cells make this type of transformation possible. For this transformation antibiotic markers associated with foreign pieces of DNA will be used to help verify that the DNA of interest was successfully inserted into the vector.
Transformation Animation: http://www.dnai.org/b/index.html
Techniques:
1. Heat Shock Transformation
Transformation
Some bacteria take up DNA spontaneously (natural transformation), but this is not true of E. coli. Thus, artificial methods for forcing DNA entry into E. coli must be used (artificial transformation). One method for introducing DNA is a chemical transformation procedure called calcium shock. It is not known why calcium shock of E. coli induces the cells to take up DNA, but presumably it somehow loosens the cell envelope structure. This procedure works best on covalently closed circular DNA. The frequency of calcium shock transformation also decreases as the size of the plasmid increases.
Electroporation
Scientists subsequently discovered a more general procedure for artificial transformation. DNA can be forced into bacteria by exposing the bacteria to a high strength electric field. This procedure is called electroporation. Bacteria are grown to mid-exponential phase, pelleted by centrifugation and resuspended in a low ionic strength, high osmolarity solution such as 10% glycerol. DNA is added and the mixture is briefly exposed to a high strength electric field. The low ionic strength of the mixture is necessary to prevent short circuiting the electric field. The high
Learning Goals: Insert your uncut unknown plasmid into chemically competent DH-5 E.coli cells and use antibiotic resistance to confirm the success of the transformation. You should familiarize yourself with the various methods of transformation and the advantages/disadvantages of each type. You should also understand how heat shock transformation works and how chemically competent cells make this type of transformation possible. For this transformation antibiotic markers associated with foreign pieces of DNA will be used to help verify that the DNA of interest was successfully inserted into the vector.
Transformation Animation: http://www.dnai.org/b/index.html
Techniques:
1. Heat Shock Transformation
Transformation
Some bacteria take up DNA spontaneously (natural transformation), but this is not true of E. coli. Thus, artificial methods for forcing DNA entry into E. coli must be used (artificial transformation). One method for introducing DNA is a chemical transformation procedure called calcium shock. It is not known why calcium shock of E. coli induces the cells to take up DNA, but presumably it somehow loosens the cell envelope structure. This procedure works best on covalently closed circular DNA. The frequency of calcium shock transformation also decreases as the size of the plasmid increases.
Electroporation
Scientists subsequently discovered a more general procedure for artificial transformation. DNA can be forced into bacteria by exposing the bacteria to a high strength electric field. This procedure is called electroporation. Bacteria are grown to mid-exponential phase, pelleted by centrifugation and resuspended in a low ionic strength, high osmolarity solution such as 10% glycerol. DNA is added and the mixture is briefly exposed to a high strength electric field. The low ionic strength of the mixture is necessary to prevent short circuiting the electric field. The high