Difference between revisions of "20.109(S17):Purification of induced protein (Day2)"

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(→‎Part 1: Lyse BL21(DE3)pLysS pRSETb_FKBP12 cells)
(→‎Protocols)
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#*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.
 
#*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.
 
#Prepare 3 mL of lysis buffer.
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#*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: {| border="1"
+
#*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 2 mL eppendorf tube is 1.1g.
 +
#Resuspend each pellet completely in the lysis buffer.
 +
#Add lysozyme (stock concentration of 50 mg/mL) to each cell resuspension such that the final concentration is 300 μg/mL.
 +
#Incubate in the 4 °C cooler for 1 hr on the nutator.
 +
 
 +
<center>
 +
{| border="1"
 
! Stock reagent
 
! Stock reagent
 
! Final concentration of stock reagent in lysis buffer
 
! Final concentration of stock reagent in lysis buffer
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|
 
|
 
|}
 
|}
−
 
+
</center>
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#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 2 mL eppendorf tube is 1.1g.
+
−
#Resuspend each pellet completely in the lysis buffer.
+
−
#Add lysozyme (stock concentration of 50 mg/mL) to each cell resuspension such that the final concentration is 300 &mu;g/mL.
+
−
#Incubate in the 4 &deg;C cooler for 1 hr on the nutator.
+
−
 
+
 
===Part 2: Purify FKBP12 protein===
 
===Part 2: Purify FKBP12 protein===
  

Revision as of 23:57, 3 January 2017

20.109(S17): Laboratory Fundamentals of Biological Engineering

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Schedule Spring 2017        Announcements        Assignments        Homework        Communication
       1. High-throughput ligand screening        2. Gene expression engineering        3. Biomaterials engineering              

Introduction

Protocols

Part 1: Lyse BL21(DE3)pLysS pRSETb_FKBP12 cells

  1. 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.
  2. 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.
  3. Confirm your calculations with the teaching faculty, then prepare your lysis buffer solution.
  4. Weigh each cell pellet using the balance and add 1 mL of lysis buffer per g of cell pellet.
    • Note: the weight of the 2 mL eppendorf tube is 1.1g.
  5. Resuspend each pellet completely in the lysis buffer.
  6. Add lysozyme (stock concentration of 50 mg/mL) to each cell resuspension such that the final concentration is 300 μg/mL.
  7. 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

Navigation links

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