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 bottom of the tube is the purified DNA and it was placed on ice.
In vitro Transcription:
To recreate the normal cell function of transcription in vitro, we added 2 µl 10X T7 reaction buffer, 2µl ATP solution, 2µl CTP solution, 2µl GTP solution, 2µl UTP solution and 2µl T7 RNA polymerase enzyme to tubes 2 and 5 and incubated it at 37°C for 4 hours.
DNAse & RNase Treatment:
We added 21 µl nuclease-free water, 5 µl 10X digestion buffer, 2 µl DNase I, and 2 µl RNase to our tubes 2 and 5. We flicked the tube and centrifuged the tubes. The tubes were placed in water bath heated to 37°C for 30 minutes. After the incubation period, the tubes were placed on ice where they waited to be added to the group electrophoresis. dsRNA Purification: