Team:Toronto/Notebook

NOTEBOOK

05/26
Prepared stock solution of LB and autoclaved empty bottles.

05/27
Prepared antibiotic aliquots and practiced pouring a gel.

05/28
Practiced dilution plating and glass bead plating. Worked collaboratively on a miniprep and used Nanodrop to evaluate the DNA purity.
Met with Michael to talk about lambda red recombination. He is trying to make his own deletion strains with a pKD46 plasmid, recombinases, and PCR. Researched plasmid map of pBR322 to determine key characteristics like antibiotic resistance, origin of replication etc.

05/29
Learned how to use the incubator with shaker and how to operate the Moraes lab Thermocycler and Spectrophotometer (details uploaded to Wiki). Also investigated the ideal rbs sequence for our genes. According to From Genes to Clones, the rbs should be 4-9bp upstream of ATG. It's stronger if you add adenines upstream of the RBS and it comes before minor genes in an operon. If we're interested in operon expression we must keep the stoichiometric balance of each minor gene by preserving the native rbs sequences.

05/30
Project Milestones are established:
1. Primer design with pMal vector.
2. Amplification and cloning of genes
3. Establishing selection and cultivation conditions with assays and stimuli
Sub-goals within each step were established as well. Prepared glycerol for inoculation and streaking. Lab members were assigned stimuli/assays to do and today we made a list of the ingredients from these protocols which we didn't have. Designed primers for CsgD after establishing RBS and other sequences.

06/01
Crystal violet assay – Discussed the different procedures for this with Afiq and Seemi.

06/02
First round of crystal violet protocols established.

06/03
Edited primers.

06/05
New cloning strategy introduced by Kristina: InFusion by Clontech. Worked to redesign primers for this purpose.15bp homology required between each insert. Prepared M63 minimal media.

06/06
Made a culture of MG1655 from a plate Michael brought over from BioZone for the purpose of making competent cells. Edited primers.

06/07
Learned to determine OD600 of growing MG culture by diluting. Made agar and 10% glycerol from 40% stock. Poured plates and streaked the little tabs that the deletions came in on top of each plate by suspending in sterile LB medium. Strains were incubated at 7 PM.

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