Difference between revisions of "20.109(S20): Prep notes for M1"
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*prepare 1% agarose gels, 1 per 2 teams | *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:''' | '''per team:''' | ||
*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 μL lysonase) |
+ | **students to add imidazole to 10 mM | ||
+ | *5 μL 2.5 M imidazole | ||
==M1D3== | ==M1D3== |
Revision as of 19:48, 5 February 2020
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, 5 μL lysonase)
- students to add imidazole to 10 mM
- 5 μL 2.5 M imidazole
M1D3
prior to laboratory:
- prepare dialysis buffer (1X PBS), 1 L per team
per team:
- 15 μL 50 mg/mL lysozyme
- lysis buffer reagents
- 170 μL 1 M Tris, pH = 7
- 470 μL 1 M NaCl
- 770 μL 40% glycerol
- 5 μL 1 M DTT
- 35 μL 10 mM AEBSF
- 1.8 mL sterile H2O
- 250 μL slurry (Ni-NTA resin) in 2 mL microcentrifuge tube
- 2.5 mL 1X PBS
- 40 μL 1 M MgCl2
- 12 μL 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:
- 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
M1D5
prior to laboratory:
- dilute 200 μM chymotrypsin (prepared in 1 mM HCl containing 2 mM CaCl2) to 2 μM chymotrypsin in H2O
- prepare reagents
- DMSO: 40% in H2O
- rapamycin: 20 μM in 40% DMSO
- ligands: 8 mM in 40% DMSO
per team:
- 18 μL 1 mg/mL FKBP12
- buffer reagents
- 1.3 mL 1 M Tris-HCl, pH = 8
- 65 μL 2 μM chymotrypsin
- 5 mL H2O
- 20 μL 40% DMSO; prepared such that 1 μL needed per reaction (final concentration = 0.2% in 200 μL)
- 10 μL 20 μM rapamycin; prepared such that 1 μL needed per reaction (final concentration = 10 μM in 200 μL)
- 10 μL 8 mM Chembridge ligands (most stocks at 100 mM); prepared such that 1 μL needed per reaction (final concentration = 40 μM in 200 μL)
during the laboratory:
- prepare 5 mM suc-AAFP-pNA in TFE containing 460 mM LiCl
M1D6
prior to laboratory:
- prepare serial dilutions of reagents (MIX WELL BETWEEN DILUTIONS)
- dye: 1000x → 100x (3 μL dye + 27 μL 1X PBS); 100x → 5x (30 μL of 100x dye + 570 μL 1X PBS)
- DMSO: 100% → 1% (5 μL DMSO + 495 μL 1X PBS)
- rapamycin: prepare 5 mM in DMSO; 5 mM → 500 μM (5 μL of 5 mM rapamycin + 45 μL 1X PBS); 500 μM → 50 μM (10 μL of 500 μM rapamycin + 90 μL 1X PBS)
- ligands: 100 mM → 20 mM (1 μL ligand in 4 μL DMSO); 20 mM → 2 mM (5 μL of 10 mM ligand + 45 μL 1X PBS); 2 mM → 200 μM (5 μL of 1 mM ligand + 45 μL 1X PBS)
per team:
- 18 μL 1 mg/mL FKBP12
- 80 μL 5x dye solution in 1X PBS (includes enough for fkbp and control protein wells)
- 24 μL 1% DMSO in 1X PBS
- 10 μL 50 μM rapamycin in 1X PBS
- 20 μL 200 μM ligand in 1X PBS
- 50 μL 1X PBS
- 35 μL Control buffer
- 55 μL sterile water
during the laboratory:
- control protein
- control ligand
- sterile H2O
- 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
per team:
- none