Project I: RNA Self-Splicing
Kit Fung (Klaus) Chan
TA: Christopher Kampmeyer, Henry Sillin
Lab Section: 1B T/R 4pm-7:50pm
Group Member: Phuong (Nhu) Huynh
Group Number: 13
Date Submitted: 4/23/2015
This is my own original work. If any portion of found not to be my own original work, I will accept zero points for this report in addition to whatever the Dean dictates. Abstract mRNA bears the role of accurately conveying genetic information from DNA into protein (Nature), but there is an extra crucial molecule between DNA and mRNA, which is the pre-mRNA. Pre-mRNA is the immature single strand of complete transcript of the DNA which contains the exon and intron. In order to become a mature mRNA, splicing has to occur to excise out the non-coding sequence (intron) and connect the coding sequences (exons) to render the correct targeted product. There are two groups in splicing reaction, in this project, the self-splicing reaction that is catalyzed by group I intron from the plasmid of Twort bacteriophage, along with the role of arginine in the self-splicing reaction will be closely examined using gel electrophoresis. The plasmid was linearized by restrictive enzyme and was loaded into a 0.8% agarose gel to confirm the presence of digested DNA. Upon confirmation, sample solution with different arginine concentration were mixed along with the digested DNA and loaded into the 2% agarose gel to confirm if self-splicing has occurred. The image from the 2% agarose gel confirmed
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With two T7 promoters, the PCR product will be transcribed by the T7 polymerase. Therefore, a double stranded RNA (dsRNA) product is produced from the plasmid, once IPTG is added to activate the lac operator of T7 polymerase.
Prior to the determination of the primary structure, the RNA strand undergoes splicing which alters the sequence. During splicing introns are removed and exons are randomly re-arranged in a random order. There is 2 problems with this model of attempting to estimate the protein a sequence will form from RNA. Firstly depending on the place of the body at which splicing is occurring, different introns will be removed and also secondly, we need to know which random order the exons will shuffle themselves into.
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coli are picked and the plasmids are purified. The purified plasmids are used as a template for the sequencing reaction. The objective of the lab was to learn how to use the polymerase chain reaction (PCR) to amplify the small subunit ribosomal RNA gene from a bacteria colony, also be able to run an agarose gel to visualize the resulting PCR amplifications and extract the amplified DNA from the agarose gel.
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