Difference between revisions of "20.109(S17):Purification of induced protein (Day2)"
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Noreen Lyell (Talk | contribs) (→Part 1: Lyse BL21(DE3)pLysS pRSETb_FKBP12 cells) |
Noreen Lyell (Talk | contribs) (→Part 1: Lyse BL21(DE3)pLysS pRSETb_FKBP12 cells) |
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Revision as of 23:44, 3 January 2017
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