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) |
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#Confirm your calculations with the teaching faculty, then prepare your lysis buffer solution. | #Confirm your calculations with the teaching faculty, then prepare your lysis buffer solution. | ||
#Weigh each cell pellet using the balance and add 1 mL of lysis buffer per g of cell pellet. | #Weigh each cell pellet using the balance and add 1 mL of lysis buffer per g of cell pellet. | ||
| − | #*Note: the weight of the | + | #*Note: the weight of the 50 mL conical tube is XX g. |
| − | #Resuspend each pellet completely in the lysis buffer. | + | #Resuspend each pellet completely in the lysis buffer then transfer the cell suspension to a 2 mL eppendorf tube. |
| − | #Add lysozyme (stock concentration of 50 mg/mL) to each cell | + | #Add lysozyme (stock concentration of 50 mg/mL) to each cell suspension such that the final concentration is 300 μg/mL. |
#Incubate in the 4 °C cooler for 1 hr on the nutator. | #Incubate in the 4 °C cooler for 1 hr on the nutator. | ||
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===Part 2: Purify FKBP12 protein=== | ===Part 2: Purify FKBP12 protein=== | ||
Revision as of 00:10, 4 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.
- Confirm your calculations with the teaching faculty, then prepare your lysis buffer solution.
- Weigh each cell pellet using the balance and add 1 mL of lysis buffer per g of cell pellet.
- Note: the weight of the 50 mL conical tube is XX g.
- Resuspend each pellet completely in the lysis buffer then transfer the cell suspension to a 2 mL eppendorf tube.
- Add lysozyme (stock concentration of 50 mg/mL) to each cell suspension such that the final concentration is 300 μg/mL.
- Incubate in the 4 °C cooler for 1 hr on the nutator.
| 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 | |
| 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