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 …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 …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