RFP Lab
Procedure: The purpose of this lab was to show the Red Flourescent Protein that is made in jellyfish.
Materials: Lab 2A - materials and procedure can be found in Amgen lab manual 2a Lab 4A - materials and procedure can be found in Amgen lab manual 4a Lab 6A - materials and procedure can be found in Amgen lab manual 6a Expirimental Overview: Lab 2A: Verification of Plasmid by Restriction Digest - In this part of the lab, we cut a plasmid (a circular piece of DNA) with BamH1 and HindIII. In doing so, we were able to extract the segment of the RFP-Ara gene. Lab 4A: Verification of Plasmid Digest by Electrophoresis - To ensure that we had actually cut the plasmid correctly, we used the process of electrophoresis. This means that we ran the plasmid in a gel alongside a DNA ladder. A DNA ladder has different-sized molecules already programmed into it, so after being run, it provides a "ladder" of bands that can be corresponded with the other lanes in order to decipher their molecule sizes. Because we already knew the size of the RFP gene and the plasmid, we were able to say if the digest had gone smoothly. Lab 5A: Transformation of Bacteria with Recombinant Plasmid - The heart of the RFP lab was in this section, where we transformed the bacteria into a recombinant (man-made) plasmid. We used restriction enzymes to cut the plasmid and ligase to paste the gene of interest. We then used a selective marker (Amp-R, resistance to Ampicillin). Lab 6A: Separating the RFP Gene with Column Chromatography - After incubating the RFP gene, we used a chromatography column in order to separate the RFP from everything else. We did this by using a column with hydrophilic beads: this effectively collected the RFP in the resin bed. Then we eluted the RFP and knocked it off those beads by using a buffer. Data Results: |