Objective:
We started by isolating a DNA plasmid from a bacterial culture for cloning. Then, we reverse transcribed mRNA to amplify it in a polymerase chain reaction. Finally, we amplified a specific gene (actin) through PCR.
Protocol:
A. Isolation of Plasmid DNA from a Bacterial Culture:
We were provided with a tube containing 1 ml of bacterial culture. We centrifuged it for 5 minutes at 10000 g. Then, we removed the supernatant by pouring it off and we used a small chem wipe to remove any excess media remained. Centrifuging will form a small pellet at the bottom of the tube. Using 250 l of cell resuspension solution, resuspend the cell pellet thoroughly by pipetting or vortexing. This will allow the cell lysis solution, which will be added next, to reach all cells. Then, add cell lysis solution to break the membranes, lyse cells, and release all the components of the cell in the tube. Invert the tube four time to mix all the components of the tube. However, do not shake vigorously or pipette or vortex the eppendorf tube because that may denature the DNA. After the cell is lysed, some proteins will be produced, which can have a negative impact of the quality of the isolated DNA. Therefore, you must add 10 l of alkaline protease solution to denature all the proteins. Invert the tube couple of times and incubate at room temperature. Make sure that you do not leave the alkaline protease solution in the tube for more than 5 minutes because it can start denaturing DNA. Then, neutralize all cells by adding 350 l neutralization solution and centrifuge lysate at maximum speed for 10 minutes at room temperature. After centrifuging is complete, transfer clear lysate to spin column without transferring the white precipitate. If the white precipitate is transferred to the spin column, pour the contents to a sterile 1.5 ml micro-centrifuge tube and centrifuge for 5-10 minutes at maximum speed. Then, transfer the supernatant into the initial spin column. After transferring the cleared lysate to a spin column, incubate at room temperature for 5-10 minutes to allow DNA binding to the membrane. Then, centrifuge the supernatant at maximum speed for about 1 minute at room temperature. Discard the flowthrough from the collection tube. In order to start washing, add 750 l column wash solution diluted with ethanol. Centrifuge at maximum speed for 1 minute at room temperature. Remove the flowthrough from the spin columns and discard it. Wash again with 250 l column wash solution and centrifuge for about a minute at maximum speed at room temperature. Discard wash and wash centrifuge again at maximum speed for 2 minutes. Transfer spin column to a new sterile 1.5 ml tube and add 50 l of nuclease free water to membrane to elute plasmid. Finally, incubate at room temperature, centrifuge for 1 minute at maximum speed, and store at -20 C. At the end of this lab, you should measure the …show more content…
Reverse Transcription of mRNA and Designing Primers for PCR:
In this experiment, we will start by preparing the RNA template, the primer, and the reverse transcriptase. The primer should bind to the RNA template and the reverse transcriptase will then be annealed to the primer and it will start elongating the cDNA strand based on the original RNA template. In each part we prepared three different tubes: 1) RT positive control, 2) RT negative control, and 3) RNA negative control. The purpose of preparing 3 different tubes is to ensure that our product is not contaminated and also to confirm that we are getting the right bands, when we run our product through the gel.
Part a: Preparation of RNA tubes:
First, you have to ensure that all your reagents and tubes are on ice because a minor change in temperature can greatly impact the reagents and products. In the RNA+RT tube, add 1 l RNA, 1 l random primer, and 3 l nuclease free water to mix RNA and RT. In the RNA–RT tube, add 1 l RNA, 1 l random primer, and 3 l nuclease free water. In the RNA–RNA tube, 1 l random primer and 4 l nuclease free water. The total volume of each tube should be 5 l. Then, start incubating the three tubes at 70 C for 5 minutes to denature 2 structures within the RNA. Finally, place the three tubes in iced water for at least 5 minutes and spin the tubes for 30 seconds to collect all the components and leave on
We started by isolating a DNA plasmid from a bacterial culture for cloning. Then, we reverse transcribed mRNA to amplify it in a polymerase chain reaction. Finally, we amplified a specific gene (actin) through PCR.
Protocol:
A. Isolation of Plasmid DNA from a Bacterial Culture:
We were provided with a tube containing 1 ml of bacterial culture. We centrifuged it for 5 minutes at 10000 g. Then, we removed the supernatant by pouring it off and we used a small chem wipe to remove any excess media remained. Centrifuging will form a small pellet at the bottom of the tube. Using 250 l of cell resuspension solution, resuspend the cell pellet thoroughly by pipetting or vortexing. This will allow the cell lysis solution, which will be added next, to reach all cells. Then, add cell lysis solution to break the membranes, lyse cells, and release all the components of the cell in the tube. Invert the tube four time to mix all the components of the tube. However, do not shake vigorously or pipette or vortex the eppendorf tube because that may denature the DNA. After the cell is lysed, some proteins will be produced, which can have a negative impact of the quality of the isolated DNA. Therefore, you must add 10 l of alkaline protease solution to denature all the proteins. Invert the tube couple of times and incubate at room temperature. Make sure that you do not leave the alkaline protease solution in the tube for more than 5 minutes because it can start denaturing DNA. Then, neutralize all cells by adding 350 l neutralization solution and centrifuge lysate at maximum speed for 10 minutes at room temperature. After centrifuging is complete, transfer clear lysate to spin column without transferring the white precipitate. If the white precipitate is transferred to the spin column, pour the contents to a sterile 1.5 ml micro-centrifuge tube and centrifuge for 5-10 minutes at maximum speed. Then, transfer the supernatant into the initial spin column. After transferring the cleared lysate to a spin column, incubate at room temperature for 5-10 minutes to allow DNA binding to the membrane. Then, centrifuge the supernatant at maximum speed for about 1 minute at room temperature. Discard the flowthrough from the collection tube. In order to start washing, add 750 l column wash solution diluted with ethanol. Centrifuge at maximum speed for 1 minute at room temperature. Remove the flowthrough from the spin columns and discard it. Wash again with 250 l column wash solution and centrifuge for about a minute at maximum speed at room temperature. Discard wash and wash centrifuge again at maximum speed for 2 minutes. Transfer spin column to a new sterile 1.5 ml tube and add 50 l of nuclease free water to membrane to elute plasmid. Finally, incubate at room temperature, centrifuge for 1 minute at maximum speed, and store at -20 C. At the end of this lab, you should measure the …show more content…
Reverse Transcription of mRNA and Designing Primers for PCR:
In this experiment, we will start by preparing the RNA template, the primer, and the reverse transcriptase. The primer should bind to the RNA template and the reverse transcriptase will then be annealed to the primer and it will start elongating the cDNA strand based on the original RNA template. In each part we prepared three different tubes: 1) RT positive control, 2) RT negative control, and 3) RNA negative control. The purpose of preparing 3 different tubes is to ensure that our product is not contaminated and also to confirm that we are getting the right bands, when we run our product through the gel.
Part a: Preparation of RNA tubes:
First, you have to ensure that all your reagents and tubes are on ice because a minor change in temperature can greatly impact the reagents and products. In the RNA+RT tube, add 1 l RNA, 1 l random primer, and 3 l nuclease free water to mix RNA and RT. In the RNA–RT tube, add 1 l RNA, 1 l random primer, and 3 l nuclease free water. In the RNA–RNA tube, 1 l random primer and 4 l nuclease free water. The total volume of each tube should be 5 l. Then, start incubating the three tubes at 70 C for 5 minutes to denature 2 structures within the RNA. Finally, place the three tubes in iced water for at least 5 minutes and spin the tubes for 30 seconds to collect all the components and leave on