Difference between revisions of "Team:Uppsala/Notebook"
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Revision as of 14:35, 31 October 2017
Week: 07/06-16/06
- A safety demonstration was done by the Lab technicians and Project managers which enabled us to know the fire escapes, first aid and emergency contacts. Lab space was assigned.
- Buffers and stock solutions were prepared. LB media and LB-agar plates with different antibiotics (Kanamycin, Chloramphenicol and Tetracycline) were prepared.
- E. coli DH5α competent cells were prepared. Ordered constructs for the three genes (CCD, ADH and UGT) were digested with available enzyme (E and S) and ligated to Linearized plasmid backbones from the iGEM Kit.
- Prepared competent cells were tested with the iGEM Competent Cells Test kit. No growth was observed. Possible causes: Faulty competent cells, improper digestion or transformation protocol.
- Made DH5α competent cells with another new protocol to be tested and used later.
Week: 19/06- 22/06
- Testing of the competent cells made last week was done with the competent cells test kit. There was growth suggesting that competent cells were working good.
- To test if the restriction digestion, ligation and transformation worked well, they were repeated again with the newly prepared and tested competent cells above. No growth was observed even when high concentrations were plated, suggesting that the fault was in the Restriction digestion, ligation steps
Week: 26/06-30/06
- On expert suggestion, it was understood that the enzymes need few extra bases on the ends for efficient activity. Thus we designed and ordered primers that would add overhangs to our constructs. Other reason could be that the promoters were too strong and the genes were toxic to the cell. So a set of primers were also designed to remove promoter from between the prefix and RBS.
- To circumvent the high toxicity of the gene, we decided to clone the devices in low copy plasmids. For that low copy plasmids were identified from the master strain database of glycerol stock at the lab, grown overnight in agar plates, grown overnight in LB and plasmids were purified.
- Digestion was tested with X and S to optimize the protocol for digestion
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