Experiment Protocols&Notebook

July

Week1(0710-0714) - “constructing pET28a-GST-MST2 1-308 and pET28a-HST-STRN3 64-145 vector”

-Prepare the following reaction system (PCR)

-PCR

-DNA Gel Electrophoresis

-DNA extract from gel

-Preparation of competent cells (CaCl2 method)

-Heat Shock Transformation

-TB liquid Culture Medium

- LB Culture Medium

- Plasmids extraction

Week2(0717-0721) - “ Expression and purification of proteins”

-Inducing protein expressions

-Protein extraction

-Staining and decolorisation with Caumas Brilliant Blue

-Protein purification

-Western Blotting

Week3(0724-0728) – “High-throughput screening by Alphascreen”“ Pull-down” “Virtual docking (in software ‘autodock’)”

-High-throughput screening by Alphascreen

-Pull-down

-Virtual docking (in software ‘autodock’)

August

Week4(0731-0804) – “constructing pET28a-GST-MST2 1-308 mutant vector”

-Sequencing of mutated gene

-Mass of amplification of bacteria

-Inducing protein expressions

-Protein purification

Week5(0807-0811) –“Pull-down” “Cell culture and CX6258 molecular medicine test” “Model animals fed with drugs”

-Protein examination

-Negative control group

-Binding

-CX test sample preparation

-Preparation of negative control and positive control

-Pull-down

- Feeding Drosophila with CX6258 drug

Week6(0814-0818) – “Observe Cell drug testing” “Analysis animal model drug testing”

-Cell clonal formation

-Use crystal violet to dye the cells

-Count the amount of the cells

-Drosophila intestinal anatomy

-Immunofluorescence staining of Drosophila intestinal tissue