Overview · Ligated fragments coding for sfGFP and mNeonGreen into YEp352 vectors · Roughly confirmed presence of fragment through agarose gel · Performed transformation of S. cerevisiae BY4741 with plasmids roughly confirmed to contain our biobricks
· Digested YEp352 vectors were ligated with either our sfGFP biobrick or our mNeon Green biobrick after PCR cleanup to remove one of the enzymes that was not heat killed · Samples of sfGFP ligated vectors were ran in parallel to mNeon Green ligated vectors · The ligation product was transformed into E. Coli for cloning · Cells were mini-prepped to extract our plasmid · To confirm the extracted sample consisted on plasmid containing our fragment of interest, a gel was run · To prepare for the gel check, a portion of the mini-prep product was digested · The enzyme should cleave the plasmid on either side of the previously ligated fragment, resulting in two pieces of DNA, the cut vector (approx. 5kb) and the naked fragment (approx. 2 kb) · Figure 2 shows a clear line at 5kb and faint bands at 2kb, indicating that the fragments should be contained with mini-prepped plasmid
· We used common wet lab procedures requiring common wet lab equipment. · All protocols are listed under the Experiment tab and provide clear instructions for those interested in replicating our work.