Team:UCC Ireland/Project/Notebook/14/08
Monday-14/08/17
- Resuspended primers
- Diluted primers down to a concentration of 10microM
- Made up fresh M9, LB agar and LB broth.
- Made fresh Cam and Amp plates.
- Plated more Church plasmid cultures and ligated Tet cultures.
- PCRed up Erythromycin, Mdh and FrmR g-blocks with primers to add correct adapters.
- Ran a gel to confirm that the PCR worked successfully.
- Received PRSF-duet backbone.
- Visited Blackwater gin microdistillery in Waterford
Tuesday-15/08/17
- Restriction digestion of the Prsf-duet backbone with Acl1 enzyme.
- Ran a gel to confirm the digestion worked
- New primers designed and ordered.
Wednesday-16/08/17
- Set up the growth curve experiment using ligated TetR-RFP and Church plasmid. Put in plate reader overnight shaking.
- Ran the PCR experiment of the gblocks a final time - Strange results?? - Confirmed that primers are incorrect.
- Made Tetracycline plates
Thursday-17/08/17
- Growth curve experiment examined. Here are the results:
- Experiment testing the OD of the amilCP cells at T0, T1.5, T3, T4.5, T6. Cells left shaking in a flask. 1mL taken from the flask at the various time points. From this, 300microL mixed with 2.7mL of m9 and OD measured. 10^-1, 10^-2, 10^-3 dilutions made taking from the 1mL and plated. Colonies will be counted tomorrow and number correlated with the dilution.
Friday-18/08/17
- PCRed up the iGEM linearized backbones
- Ran these samples on a gel to confirm the PCR worked
- Transformed two plasmids into DH5A E.coli via heat-shock : GFP producing plasmid and a plasmid containing a constitutive promoter. These plasmids will be ligated next week to form a constitutively producing GFP plasmid.
