The+Random+Walk

This is the path to enlightenment for VDS researchers. It may be a direct path or a random walk for you!

These are in general order - but steps can be done in parallel


 * MILESTONES**


 * Clone your Coding DNA Sequence (CDS) into expression vector**
 * Primary PCR
 * Secondary PCR
 * PCR^2
 * PCR cleanup, Nanodrop
 * Cloning protocol
 * Cut pNIC-Bsa4 (can be done once you have PCR^2 working)
 * Annealing & Transformation
 * Grow up clones
 * DNA sequencing for positives
 * Grow up positives and Midiprep, then archive


 * Virtual Screening of your target (should be done in __parallel__ with wet lab - i.e. not sequentially)**
 * Molprobity on structure
 * Create Homology Model if needed (run Molprobity on this)
 * Pick Control Ligands
 * LigPrep Control Ligands
 * Set up protein in GOLD & Dock control ligands
 * Analyze control docking
 * Dock screening libraries of novel compounds
 * Order top hits (once you have soluble protein from wet lab)


 * Express Protein**
 * Transform plasmid into expression cells
 * Express protein
 * Harvest & Purify protein through Nickel affinity column and Size Exclusion (FPLC)
 * Characterize protein on gel
 * Store protein (2 ways)


 * Enzyme Assays**
 * Test out if enzyme is active (Vary enzyme)
 * Vary substrate assays (determine Km value)


 * Inhibition Assays**
 * Test out different concentrations of compounds ordered
 * Determine IC50 if it is a good inhibitor

Virtual screening with ICM docking software Ligand based virtual screening Fluorescence melt assays Crystallization trials
 * Extra Toppings:**

carry out MM-PB(GB)SA calculations of Free Energy of Binding to re score top ligands - use Prime in Maestro Suite from Schrodinger
 * Extensions:**