Please select a week on the left.
5/29/2012 | - Designed 9 primers for LacI. LacZ. and eYFP. Prepared LB. - Inoculation of B0014.J23100.wtPUF. B0030.Q04121 - Transformed the psB1C3 biobrick into DH5a cells |
5/31/2012 | - Transformed C0012, I732005, E0030, I732017. Also transformed PSB1A3, PSB1AK3, PSB1K3. - Inoculated LB media with a psB1C3 colony from the plate grown the day before. - Met with Prof. Bhalerao to talk about PUF application and project direction (RNA scaffolding). |
6/1/2012 | - Short-term goal changed: make one testing construct, [rbs-puf-eYFP] cloned into P.protet plasmid with a ptet promoter and terminator - Began inoculation of C0012, I732005, E0030, I732017, PSB1A3, PSB1AK3, PSB1K3. Was incubated for 9 hours, then put in 4 degree room - Inoculated again due to the round-bottom tube being misplaced. Mini-prepped the inoculation. |
6/2/2012 | - Cultured [PUF+PIN], E0030, and P. protet. designed check primers for [rbs-puf-eYFP] |
6/3/2012 | - Inoculated with a psB1C3 colony in order to prepare a glycerol stock of the strain. |
6/4/2012 | - Threw out old, incomplete inoculations from Friday. - Transformed I732005, R0010. - Miniprepped Venus and E0030 and put gDNA in the -20 temp box. - Did new inoculations on C0012, E0030, I732017, PSB1A3, PSB1AK3, PSB1K3. - Made a glycerol stock of the psB1C3 strain. |
6/5/2012 | - Made glycerol stocks of C0012, E0030, I732017, PSB1A3, PSB1AK3, PSB1K3 and put them in the -80 freezer. - Transformation of I732005 failed, so replated with rest of culture. - Transformed J04500 and PSB3T5. - Minipreped PUF Amp and protet CM20 (spilled 1 tube of this) and put gDNA in the -20 degree in the temp box. - Inoculated R0010. |
6/7/2012 | - Set goal to create RNA scaffold by 6/15, begun design. |
6/11/2012 | - Innoculated K12 - Transformed split GFP constructs BBa_K157005 and BBa_K157006 into DH5a cells - Constructed the first RNA scaffold draft - Inoculated both parts of split GFP, as well as J04500 |
6/12/2012 |
- Miniprepped K12 cells - RNA scaffold design will inculde two scaffolds which need to be stored into one plasmid and then ligated into separate plasmids - Made glycerol stock of both split GFP parts, J04500, and miniprepped both split GFP |
6/13/2012 | - Transformed Pertobrick into K12 cells - Constructed the first draft of the scaffold, but it needs review. - Second, unique scaffold needs to be designed |
6/14/2012 | - Plated Petrobrick transformations; inoculated cell growth using CM resistance - Finalized an updated version of the scaffold based on new data of PUF binding. Final modifications need to be made before sent for synthesis. - Performed PCR: (2) 50 uL tubes of control (no PUF binding site) and (2) 50 uL tubes with the PUF binding site. Used WT PUF as a template and phusion as the polymerase. Ran gel and saw a result of all primer dimers. |
6/15/2012 | - Made plan to digest cells in order to see whether or not petrobrick was transformed into K12. - Final modifications made on the scaffold design. The construct is ready for synthesis. - Second try of PCR: Did a temperature gradient of 55-63-71 degrees Celsius. Used the WT PUF as template DNA. Gel results show only primer dimers, the faintest of faint lines at around 700 base pairs, but it's not a positive result. - Miniprepped pTET is in the "temporary" box of the -20 degree Celsius freezer. |