6/4/14 Objective: Prepare buffers and mediums for new CCEC protocol Matthew Farnitano, TJ Ciesla, Mike Zhu, Charlie Cooper Prepare SOB Medium for bacterial transformation Protocol from iGEM’s parts website http://parts.igem.org/Help:Protocols/Competent_Cells Made 1 L, autoclaved and stored in two 500 mL containers in cold room medium still appeared cloudy before autoclave--may just be new recipe Prepare CCMB 80 Buffer for making chemically competent E. coli cells Protocol from iGEM’s parts website http://parts.igem.org/Help:Protocols/Competent_Cells Made 1 L, filtered and stored in two 500 mL containers in cold room pH 6.34 (overshot a few times pH adjustment, but no noticeable precipitate formed Autoclave two 500 mL culture flasks For CCEC protocol With water inside to remove detergent residues Next steps: Prepare CCEC Objective: Attempt to grow some transformed cell cultures with Charlie’s CCEC Matthew Faw The second (and more properly conducted) attempt to transform the CCEC with RFP construct BBa_J04450 from 6/2 failed. Today, we are trying to see if we can get any transformed cells to grow in plates. Followed Charlie’s Cloning protocols,with slight modifications Added 5 lof 0 (control) and 50pg/lRFP Construct BBa_J04450 to Charlie’s chemically competent E. Coli, and followed procedure Plated the transformed DNA on 2 separate plates, put in put in 37C overnight Results: No colonies grew (6/5/14) Next Steps: -Examine plates to see if any cultures grew -Attempt the transformation with the cell cultures growns using iGEM’s standard protocols that can be found here: http://parts.igem.org/Help:Protocols/Competent_Cells -Lab currently in the process of making these CCEC Objective: Prepare pdCas9 marrafini and pCsy4 Arkin plasmids to be miniprepped Matthew Faw, Charlie Cooper Prepare pdCas9 and pCsy4 to be miniprepped tomorrow Put pdCas9 into a culture tube with 5ml SOC+Chloramphenicol Put pCsy4 in a culture tube with 5ml SOC+Amp Left both to shake at 37C to grow the plasmid DNA--Charlie will retrieve them Next steps: Miniprep plasmid DNA