Utk knoxville may

May 18, 2012: Primers designed and ordered
May 20, 2012: Morgan and Akshitha made a plan for cloning
May 22, 2012: Primers received and resuspended
May 24, 2012: Morgan transformed mORANGE from Plate 2, Well 13N (BBa_E2050) into Top10 chemically competent cells. Akshitha transformed backbone (BBa_J63010) into Top10 chemically competent cells.
May 25, 2012: Morgan inoculated two colonies from each of the transformed plates into liquid medium for plasmid extraction.
- Also inoculated colonies from BBa_17107 plate that Michael already had for GFP.
May 26, 2012: Morgan performed a plasmid extraction of mORANGE and GFP plasmids.
- Akshitha plasmid extrancted BBa_J63010.
- She also ran a PCR to amplify out mORANGE, GFP, and ADH genes.
- mORANGE and GFP worked. They were extracted from gel. Adh did not amplify. Tried again using different annealing temps(63,68,73,78). Did not work. Tried again using a different template (PRS426Adh in adam’s box) and lower annealing temps (45, 50, 55)
- She digested all three (plus backbone) and ligated to form constitutively expressed fluorescent constructs for our controls.