20.109(S17):Purification of induced protein (Day2)
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Contents
Introduction
Protocols
Part 1: Lyse BL21(DE3)pLysS pRSETb_FKBP12 cells
- Retrieve your BL21(DE3)pLysS pRSETb_FKBP12 cell pellet from the front laboratory bench and leave them on your bench to thaw.
- You will also obtain a cell pellet of uninduced cells that was prepared by the teaching faculty as a control to examine the IPTG induction step.
- Prepare 3 mL of lysis buffer.
- Use the information in the table below to calculate the volume of each stock reagent that is needed to prepare the lysis buffer with the specified final concentrations:
Stock reagent | Final concentration of stock reagent in lysis buffer | Volume of stock reagant |
---|---|---|
1 M Tris (pH = 7) | 50 mM | |
1 M NaCl | 150 mM | |
40% glycerol | 10% | |
1 M DTT | 1 mM | |
1 M AEBSF | 1 mM | |
50 mg/mL lysozyme | 300 μg/mL | |
H2O | add for a total of 3 mL of lysis buffer |
Part 2: Purify FKBP12 protein
Reagents
Next day: Evaluation of purified protein
Previous day: In silico cloning and induction of protein expression