DNA purification: To get pure DNA to use, a silica membrane was used to collect the long DNA strands. We started by adding 5 volumes of PB buffer to 1 volume of each PCR reaction. We put column and the silica wafers into tubs and pipetted the DNA samples onto the wafers. We centrifuged the tubes for a minute and disposed of the waste. After that we pipetted 0.75 ml of buffer PE to the samples and spun it for another minute then disposed of the waste. The columns were added to the tubes and spun for a minute in the centrifuge to dry then planed in new clean tubes. Without touching the wafers with the pipet tip, 50µl of elution buffer EB was added to the columns then left for a minute and centrifuged for a minute. The liquid collected at the
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The entire (500 µl) mixture was pipetted onto a purification column and spun for 2 minutes in the centrifuge. The filtered waste was disposed of and the column was placed into the tube. 500 µl of wash buffer was added to the column then spun in the centrifuge for another two minutes and the waste was put in the waste container. This washing procedure was repeated once more. After tossing the waste on the second cycle, the column was spun to dry and then transfer to a clean tube labelled with the date and Thread or DRP1. We added 100 µl of elution solution on top of the column and incubated tube for 2 minutes at 69°C. After the incubation period, the tube was centrifuged for 3 minutes. The liquid collected in the tube contains the dsRNA and was put on ice to await the next …show more content…
For the DRP1 samples we set up tubes labelled A, B, and C. Tube A contained 249.5 µl of serum free media and 0.5 µl Mito-GFP plasmid. Tube B contained 224.5 µl serum free media, 0.5 µl mito-GFP plasmid, and 25 µl DRP1 dsRNA. Two tube C were premade and contained 240 µl and 10 µl cellfection liposome solution. After the tubes were mixed by hand, 250 µl of tube C was added to A and B and mixed gently before being incubated for 30 minutes.
Examining Phenotypes and Microscopy: To examine the results of thread manipulation, we captured images of the control and affected Thread cells. We used a light microscope with an HDMI port attached to a computer. We photographed both controlled and affected samples three times each. The slide images were saved onto the laptop and sent to the group member’s emails.
To examine the results from the DRP1 gene inhibition, the cells had to be injected with mito-GFP. Mito-GFP is a green fluoresces gene from jellyfish. The GFP insertion allowed us to examine the mitochondria in the cells with a fluorescence microscope. This microscope takes three versions of the image and we took three different images from both the dsRNA DRP1 and controlled samples. The images were saved onto the computer and uploaded onto the lab’s Blackboard
The entire (500 µl) mixture was pipetted onto a purification column and spun for 2 minutes in the centrifuge. The filtered waste was disposed of and the column was placed into the tube. 500 µl of wash buffer was added to the column then spun in the centrifuge for another two minutes and the waste was put in the waste container. This washing procedure was repeated once more. After tossing the waste on the second cycle, the column was spun to dry and then transfer to a clean tube labelled with the date and Thread or DRP1. We added 100 µl of elution solution on top of the column and incubated tube for 2 minutes at 69°C. After the incubation period, the tube was centrifuged for 3 minutes. The liquid collected in the tube contains the dsRNA and was put on ice to await the next …show more content…
For the DRP1 samples we set up tubes labelled A, B, and C. Tube A contained 249.5 µl of serum free media and 0.5 µl Mito-GFP plasmid. Tube B contained 224.5 µl serum free media, 0.5 µl mito-GFP plasmid, and 25 µl DRP1 dsRNA. Two tube C were premade and contained 240 µl and 10 µl cellfection liposome solution. After the tubes were mixed by hand, 250 µl of tube C was added to A and B and mixed gently before being incubated for 30 minutes.
Examining Phenotypes and Microscopy: To examine the results of thread manipulation, we captured images of the control and affected Thread cells. We used a light microscope with an HDMI port attached to a computer. We photographed both controlled and affected samples three times each. The slide images were saved onto the laptop and sent to the group member’s emails.
To examine the results from the DRP1 gene inhibition, the cells had to be injected with mito-GFP. Mito-GFP is a green fluoresces gene from jellyfish. The GFP insertion allowed us to examine the mitochondria in the cells with a fluorescence microscope. This microscope takes three versions of the image and we took three different images from both the dsRNA DRP1 and controlled samples. The images were saved onto the computer and uploaded onto the lab’s Blackboard