Purification and Quantitation of DNA and Plasmid Structure
Labs 3 and 4
Biology 453
Clayton Pedigo
September 14 and 21, 2016
Introduction:
In this report, there are two labs being discussed. The first lab being lab 3 (purification and quantitation of plasmid DNA) involved lysing bacterial E.coli cells and retrieving the plasmid particles. The purpose of the second lab, lab 4 (plasmid DNA structure), was to use the concentrated DNA from lab 3 and compare the electrophoretic migrations of the different forms of the plasmid. Plasmids are circular genetic structures in a cell that can be used to replicate DNA independently of the chromosomes. The use of many different bacterial cells containing plasmids …show more content…
The cells were centrifuged for two minutes at maximum speed. Once centrifugation is completed the supernatant was removed and 250 microliters of re-suspension (R3) was added to the remaining bacterial pellet. This was then re-suspended using a pipet, until homogeneous. Then 250 microliters of lysis solution was added. This solution contains SDS detergent, which dissolves the cell membrane and denatures proteins. Then the tube was gently mixed by inverting four to six times. Following the inversion, 350 microliters of precipitation buffer (N4) was added and the solution was inverted until the mixture is homogenous. Then it was centrifuged at full speed for five minutes. The supernatant was transferred to a mini spin column. Then this column was centrifuged for one minute at full speed. The flow through was discarded. Then 750 microliters of wash buffer (W9) was added to the column and it was centrifuged for one minute at 12,000 rpm. The flow through was removed and tube was centrifuged again to remove any left over wash solution. Then the spin column was placed in a clean 1.5-milliliter micro centrifuge tube and was incubated for one minute at room temperature. 50 microliters of elution buffer (E1) was then added to the center of the column and the tube was incubated at room temperature for one minute. Then the column was centrifuged for one minute at full
Labs 3 and 4
Biology 453
Clayton Pedigo
September 14 and 21, 2016
Introduction:
In this report, there are two labs being discussed. The first lab being lab 3 (purification and quantitation of plasmid DNA) involved lysing bacterial E.coli cells and retrieving the plasmid particles. The purpose of the second lab, lab 4 (plasmid DNA structure), was to use the concentrated DNA from lab 3 and compare the electrophoretic migrations of the different forms of the plasmid. Plasmids are circular genetic structures in a cell that can be used to replicate DNA independently of the chromosomes. The use of many different bacterial cells containing plasmids …show more content…
The cells were centrifuged for two minutes at maximum speed. Once centrifugation is completed the supernatant was removed and 250 microliters of re-suspension (R3) was added to the remaining bacterial pellet. This was then re-suspended using a pipet, until homogeneous. Then 250 microliters of lysis solution was added. This solution contains SDS detergent, which dissolves the cell membrane and denatures proteins. Then the tube was gently mixed by inverting four to six times. Following the inversion, 350 microliters of precipitation buffer (N4) was added and the solution was inverted until the mixture is homogenous. Then it was centrifuged at full speed for five minutes. The supernatant was transferred to a mini spin column. Then this column was centrifuged for one minute at full speed. The flow through was discarded. Then 750 microliters of wash buffer (W9) was added to the column and it was centrifuged for one minute at 12,000 rpm. The flow through was removed and tube was centrifuged again to remove any left over wash solution. Then the spin column was placed in a clean 1.5-milliliter micro centrifuge tube and was incubated for one minute at room temperature. 50 microliters of elution buffer (E1) was then added to the center of the column and the tube was incubated at room temperature for one minute. Then the column was centrifuged for one minute at full