Difference between revisions of "20.109(S20): Prep notes for M1"

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(→‎M1D2)
(→‎M1D2)
 
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*25 μL 6x loading dye
 
*25 μL 6x loading dye
−
*3 mL lysis buffer: 50 mM NaH<sub>2</sub>PO<sub>4</sub>, 300 mM NaCl, 5 &mu;L lysonase)
+
*3 mL lysis buffer: 50 mM NaH<sub>2</sub>PO<sub>4</sub>, 300 mM NaCl
−
**students to add imidazole to 10 mM
+
**students to add imidazole to 10 mM and 5 &mu;L lysonase
−
*5 &mu;L 2.5 M imidazole
+
*15 &mu;L 2.5 M imidazole
 +
*500 &mu;L 50% nickel-resin (Ni-NTA) slurry
 +
*1.2 mL 1X PBS
 +
*8 mL 10 mM imidazole in 1X PBS
 +
*3 mL HRV 3C buffer
  
−
==M1D3==
 
−
'''prior to laboratory:'''
 
−
*prepare dialysis buffer (1X PBS), 1 L per team
 
  
−
'''per team:'''
+
*front laboratory bench: lysonase, SnapTag substrate, DTT, HRV 3C protease
  
−
*15 &mu;L 50 mg/mL lysozyme
+
==M1D3==
−
*lysis buffer reagents
+
−
**170 &mu;L 1 M Tris, pH = 7
+
−
**470 &mu;L 1 M NaCl
+
−
**770 &mu;L 40% glycerol
+
−
**5 &mu;L 1 M DTT
+
−
**35 &mu;L 10 mM AEBSF
+
−
**1.8 mL sterile H<sub>2</sub>O
+
−
*250 &mu;L slurry (Ni-NTA resin) in 2 mL microcentrifuge tube
+
−
*2.5 mL 1X PBS
+
−
*40 &mu;L 1 M MgCl<sub>2</sub>
+
−
*12 &mu;L DNase
+
−
*5 mL 1X PBS containing 10 mM imidazole
+
−
*3.5 mL 1X PBS containing 250 mM imidazole
+
−
 
+
−
 
+
−
2/12/18 Calculations for 10 aliquots of Imidazole, make fresh the day of:
+
−
*250mM Imidazole: 0.68 g of Imidazole in 40 mL 1X PBS
+
−
*10mM Imidazole: 2 mL of 250mM stock + 48 mL 1X PBS
+
−
 
+
−
==M1D4==
+
 
'''prior to laboratory:'''
 
'''prior to laboratory:'''
  
Line 72: Line 53:
 
**400 &mu;L BCA Reagent B
 
**400 &mu;L BCA Reagent B
  
−
==M1D5==
+
==M1D4==
−
'''prior to laboratory:'''
+
'''per team:'''
  
−
*dilute 200 &mu;M chymotrypsin (prepared in 1 mM HCl containing 2 mM CaCl<sub>2</sub>) to 2 &mu;M chymotrypsin in H<sub>2</sub>O
+
*12 mL TBS-T
−
*prepare reagents
+
*4 mL TBS
−
**DMSO: 40% in H<sub>2</sub>O
+
−
**rapamycin: 20 &mu;M in 40% DMSO
+
−
**ligands: 8 mM in 40% DMSO
+
  
−
'''per team:'''
 
  
−
*18 &mu;L 1 mg/mL FKBP12
+
*front laboratory bench: dH<sub>2</sub>O reservoirs for 'dunking' slides
−
*buffer reagents
+
−
**1.3 mL 1 M Tris-HCl, pH = 8
+
−
**65 &mu;L 2 &mu;M chymotrypsin
+
−
**5 mL H<sub>2</sub>O
+
−
*20 &mu;L 40% DMSO; prepared such that 1 &mu;L needed per reaction (final concentration = 0.2% in 200 &mu;L)
+
−
*10 &mu;L 20 &mu;M rapamycin; prepared such that 1 &mu;L needed per reaction (final concentration = 10 &mu;M in 200 &mu;L)
+
−
*10 &mu;L 8 mM Chembridge ligands (most stocks at 100 mM); prepared such that 1 &mu;L needed per reaction (final concentration = 40 &mu;M in 200 &mu;L)
+
  
−
'''during the laboratory:'''
+
==M1D5==
 +
'''per team:'''
  
−
*prepare 5 mM suc-AAFP-pNA in TFE containing 460 mM LiCl
+
*none
  
 
==M1D6==
 
==M1D6==
−
'''prior to laboratory:'''
 
−
 
−
*prepare serial dilutions of reagents (MIX WELL BETWEEN DILUTIONS)
 
−
**dye: 1000x &rarr; 100x (3 &mu;L dye + 27 &mu;L 1X PBS); 100x &rarr; 5x (30 &mu;L of 100x dye + 570 &mu;L 1X PBS)
 
−
**DMSO:  100% &rarr; 1% (5 &mu;L DMSO + 495 &mu;L 1X PBS)
 
−
**rapamycin:  prepare 5 mM in DMSO; 5 mM &rarr; 500 &mu;M (5 &mu;L of 5 mM rapamycin + 45 &mu;L 1X PBS); 500 &mu;M &rarr; 50 &mu;M (10 &mu;L of 500 &mu;M rapamycin + 90 &mu;L 1X PBS)
 
−
**ligands: 100 mM &rarr; 20 mM (1 &mu;L ligand in 4 &mu;L DMSO); 20 mM &rarr; 2 mM (5 &mu;L of 10 mM ligand + 45 &mu;L 1X PBS); 2 mM &rarr; 200 &mu;M (5 &mu;L of 1 mM ligand + 45 &mu;L 1X PBS)
 
−
 
 
'''per team:'''
 
'''per team:'''
  
−
*18 &mu;L 1 mg/mL FKBP12
+
*none
−
*80 &mu;L 5x dye solution in 1X PBS (includes enough for fkbp and control protein wells)
+
−
*24 &mu;L 1% DMSO in 1X PBS
+
−
*10 &mu;L 50 &mu;M rapamycin in 1X PBS
+
−
*20 &mu;L 200 &mu;M ligand in 1X PBS
+
−
*50 &mu;L 1X PBS
+
−
*35 &mu;L Control buffer
+
−
*55 &mu;L sterile water
+
−
 
+
−
 
+
−
'''during the laboratory:'''
+
−
*control protein
+
−
*control ligand
+
−
*sterile H<sub>2</sub>O
+
−
*filtered pipet tips
+
−
 
+
−
'''Bring to Koehler lab'''
+
−
*control protein (2 ul/well, 6.5 uL/mastermix)
+
−
*FKBP12 (ab85840, 1.2uL/well, 3.9 uL/mastermix)
+
−
*plates & seals
+
−
*Pipettes & tips
+
  
 
==M1D7==
 
==M1D7==

Latest revision as of 20:35, 5 February 2020

20.109(S20): Laboratory Fundamentals of Biological Engineering

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Spring 2020 schedule        FYI        Assignments        Homework        Class data        Communication
       1. Screening ligand binding        2. Measuring gene expression        3. Engineering antibodies              


M1D1

per team:

  • 25 μL pET_MBP_SNAP_TDP43-RRM12 (25 ng/uL)
  • 200 μL nuclease-free H2O

during the laboratory:

  • restriction enzymes and buffers
  • filtered pipet tips

M1D2

prior to laboratory:

  • prepare 1% agarose gels, 1 per 2 teams
  • inoculate BL21-A1 pET_MBP_SNAP_TDP43-RRM12 and induce protein expression:
    • inoculate 5 mL LB containing kanamycin with BL21-A1 pET_MBP_SNAP_TDP43-RRM12 and culture overnight
    • dilute 1:10 in 50 mL LB containing kanamycin and culture ~4 hours (until log phase)
    • add IPTG (1 mM) and arabinose (0.2%) and incubate 2.5 hr at 37 °C
    • pellet cells at 3000g for 10 min.
    • also prepare uninduced pellets

per team:

  • 25 μL 6x loading dye
  • 3 mL lysis buffer: 50 mM NaH2PO4, 300 mM NaCl
    • students to add imidazole to 10 mM and 5 μL lysonase
  • 15 μL 2.5 M imidazole
  • 500 μL 50% nickel-resin (Ni-NTA) slurry
  • 1.2 mL 1X PBS
  • 8 mL 10 mM imidazole in 1X PBS
  • 3 mL HRV 3C buffer


  • front laboratory bench: lysonase, SnapTag substrate, DTT, HRV 3C protease

M1D3

prior to laboratory:

  • boil stained and unstained molecular weight standards, if required
  • prepare electrophoresis buffer, 1 chamber volume per 2 teams

per team:

  • 20 μL Laemmli buffer
  • 50 mL coomassie stain
  • BSA reagents
    • 110 μL albumin
    • 1.3 mL 1X PBS
    • 17 mL BCA Reagent A
    • 400 μL BCA Reagent B

M1D4

per team:

  • 12 mL TBS-T
  • 4 mL TBS


  • front laboratory bench: dH2O reservoirs for 'dunking' slides

M1D5

per team:

  • none

M1D6

per team:

  • none

M1D7

per team:

  • none
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