Team:Pitt/Notebook
Notebook
Note: all experiments were done with all members present, unless otherwise noted
June 15, 2015
| Protocol | Notes/results/purpose |
|---|---|
| Transformation of plasmids from Pardee paper | Into DH5a, all transformations successful |
| Grow O/N of DH5a | Competent cell prep (Konstantin Borisov) |
June 16, 2015
| Protocol | Notes/results/purpose |
|---|---|
| O/N of transformations | Miniprep |
| DH5a competent cell prep | 25 tubes of 200ul each, enough to last all summer! (Konstantin Borisov, help from Tatyana Yatsenko) |
June 17, 2015
| Protocol | Notes/results/purpose |
|---|---|
| Transformation of plasmids from iGEM registry | Into new DH5a, all transformations successful |
June 18, 2015
| Protocol | Notes/results/purpose |
|---|---|
| Test PT7 lysate with Pardee paper plasmids | No convenient method of visualization, so we started looking for a plate reader |
June 22, 2015
| Protocol | Notes/results/purpose |
|---|---|
| PCR pSB1C3 with backbone fwd and rev primers | For Gibson assembly, learning process |
| Gel of PCR products | Visualization and purification of pSB1C3 backbone |
| Gel purification | Obtained enough backbone for the rest of the summer! |
June 23, 2015
| Protocol | Notes/results/purpose |
|---|---|
| Gibson assembly of protease parts from IDT, transformation into DH5a | All transformations multiple colonies |
| Transformation of CMU ERT7 into DH5a | Success, faint yellow from YFP seen under blue light |
June 24, 2015
| Protocol | Notes/results/purpose |
|---|---|
| Miniprep and diagnostic gel of Gibson assembly | All 3 had appropriate bands |
| Crude lysate preparation from ERT7/DH5a | Original S30 extract protocol, much too dilute due to incorrect weighing |
June 25, 2015
| Protocol | Notes/results/purpose |
|---|---|
| Sequencing of Gibson'd protease constructs | 5/6 had correct sequence, other one had significant deletion |
| ERT7 lysate tests on Tecan plate reader | No results because of high dilution |
June 29, 2015
| Protocol | Notes/results/purpose |
|---|---|
| Repetition of Pardee experiments | First success! |
| Freeze-dry constructs on paper | Test validity of Pardee results (Tatyana Yatsenko, Garrett Green) |
June 30, 2015
| Protocol | Notes/results/purpose |
|---|---|
| Grow O/N of NiCo21(DE3) | Competent cell prep (Konstantin Borisov) |
| Test freeze-dried constructs | No results on Tecan reader |
July 1, 2015
| Protocol | Notes/results/purpose |
|---|---|
| NiCo21(DE3) competent cell prep | 20 tubes of 250ul each (Konstantin Borisov) |
| Solution tests of Pardee toehold sensors | No results |
July 6, 2015
| Protocol | Notes/results/purpose |
|---|---|
| Restocked solutions, plates and media | Running low on supplies |
July 7, 2015
| Protocol | Notes/results/purpose |
|---|---|
| Transform plasmids for ERT7: wildtype T7, T7-YFP, T7 with sites | Controls for estrogen project, into DH5a |
July 9, 2015
| Protocol | Notes/results/purpose |
|---|---|
| Created cell lysates from NiCo21(DE3), NiCo21(DE3) with IPTG induction, DH5a, DH5a with ERT7-YRP | Lots of controls |
July 10, 2015
| Protocol | Notes/results/purpose |
|---|---|
| Set up PCR for vector backbone | Discovered original vector backbone had been left out and degraded |
| Prepared glycerol stocks of protease constructs | In NiCo21(DE3) cells |
July 11, 2015
| Protocol | Notes/results/purpose |
|---|---|
| Gel electrophoresis on PCR | Gel was run in the wrong direction... lost all DNA |
July 13, 2015
| Protocol | Notes/results/purpose |
|---|---|
| Another PCR of vector backbone and Gel | Success! 3 lanes of vector backbone extracted. |
July 16, 2015
| Protocol | Notes/results/purpose |
|---|---|
| Gibson of MMP9 fusion and protease reporter, transformation into DH5a | all transformations had a lot of colonies |
July 17, 2015
| Protocol | Notes/results/purpose |
|---|---|
| Diagnostic gel of all DNA made | Cut with EcoRI, SpeI (Konstantin Borisov) |
