- Reporter strain growth,
- PLATE media (PEG cannot be autoclaved and has to be filtered. A painful process. pH 8. PLATE is for PEG, Lithium Acetate, Tris and EDTA)
- Autoclave glass beads
- Prepare collection flasks
- Denatured carrier DNA on ice.
- DTT: Dithiothreitol
Section 1
By 8:14, only students are here.
Group 1 explained lab 8.1
Group 2 draw a diagram on the transformation protocol.
20 minutes lecture on review MSH2 project, transformation, selection media.
9:15, experiment started.
Changes from the protocol: Kioko gave 2ml of cells in 1 falcon tube to students. He asked students to spin down cells, add 2mL water, resuspend the cells and split them to 2 eppendorf tubes.
by 10:15am, some groups started 30C incubation. I asked students cut incubation to 10 minutes.
by 10:30, I asked to cut heat shock time to 10 minutes as well.
Kioko: DTT is a reducing agent and can break disulfide bonds on glycoprotein in cell wall and membrane, which will loosen the cell barrier for transformation.
Problems:
One group of students could identify their concentrated plasmid DNA from diluted PCR template.
Some students complained about the lack of effort on team mates.
Many students keep
Section 1 recording, https://youtu.be/AgX3odjM1vs
Section 2,
Ask group 1 to draw diagram on board.
by 1:30pm, students started diluting plasmid to 0.1ug/ul for 10 uL
Problems:
Some students insert micropipette shaft into sterile water.
Some students forgot carrier DNAs or mistook DTT as carrier DNA
Some student pour sterile glass beads into their hands and then put onto plates.
TODO: Forward and backward PCR primers
Review concepts
Genetic pathway selection
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