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Team:Brasil-SP/Notebook/Protocols
From 2014.igem.org
Experimental Methodology
For the assemblies constructuion we addopted the following method:
We used the pSB1C3 vector from the Biobrick BBa_J04450. Using this biobrick we added a red/white selection, where the red colonies are those which do not have the assembly, and the white ones are the correct ones.
Lab Protocols
- PCR
- Agarose Gel Electrophoresis and DNA Gel Purification
- PCR Product cloning
- Competent E. coli Cells using Calcium Chloride
- Transformation in E. coli DH5-alpha
- Plasmid preparation by alkaline lysis and SDS
- Cohesive-end assembly cloning
- Restriction Analysis and Digestion in large scale
- Competent Bacillus subtilis cells
- Transformation of Bacillus subtilis
- DSM medium for sporulation
- Luria-Bertani (LB) medium
- Cell counting with Neubauer Chamber
- Germination of Bacillus subtilis spores
- QuickChange PCR
- PCR for Synthesis
