Difference between revisions of "20.109(S18): Prep notes for M1"
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==M1D1== | ==M1D1== | ||
| + | '''per team:''' | ||
| + | *200 μL nuclease-free H<sub>2</sub>O | ||
| + | |||
| + | '''during the laboratory:''' | ||
*restriction enzymes and buffers | *restriction enzymes and buffers | ||
*filtered pipet tips | *filtered pipet tips | ||
| Line 18: | Line 22: | ||
*520 μL 0.1 M IPTG | *520 μL 0.1 M IPTG | ||
| − | == | + | ==M1D3== |
'''prior to laboratory:''' | '''prior to laboratory:''' | ||
*prepare dialysis buffer (1X PBS), 1 L per team | *prepare dialysis buffer (1X PBS), 1 L per team | ||
| Line 24: | Line 28: | ||
'''per team:''' | '''per team:''' | ||
| + | *15 μL 50 mg/mL lysozyme | ||
*lysis buffer reagents | *lysis buffer reagents | ||
**170 μL 1 M Tris, pH = 7 | **170 μL 1 M Tris, pH = 7 | ||
| Line 35: | Line 40: | ||
*40 μL 1 M MgCl<sub>2</sub> | *40 μL 1 M MgCl<sub>2</sub> | ||
*12 μL DNase | *12 μL DNase | ||
| − | * | + | *5 mL 1X PBS containing 10 mM imidazole |
*3.5 mL 1X PBS containing 250 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 54: | Line 64: | ||
**400 μL BCA Reagent B | **400 μL BCA Reagent B | ||
| − | == | + | ==M1D5== |
'''prior to laboratory:''' | '''prior to laboratory:''' | ||
*dilute 200 μM chymotrypsin (prepared in 1 mM HCl containing 2 mM CaCl<sub>2</sub>) to 2 μM chymotrypsin in H<sub>2</sub>O | *dilute 200 μM chymotrypsin (prepared in 1 mM HCl containing 2 mM CaCl<sub>2</sub>) to 2 μM chymotrypsin in H<sub>2</sub>O | ||
| − | *prepare | + | *prepare reagents |
| − | **DMSO: | + | **DMSO: 40% in H<sub>2</sub>O |
| − | **rapamycin: | + | **rapamycin: 20 μM in 40% DMSO |
| − | **ligands: | + | **ligands: 8 mM in 40% DMSO |
'''per team:''' | '''per team:''' | ||
| Line 67: | Line 77: | ||
*18 μL 1 mg/mL FKBP12 | *18 μL 1 mg/mL FKBP12 | ||
*buffer reagents | *buffer reagents | ||
| − | ** | + | **1.3 mL 1 M Tris-HCl, pH = 8 |
| − | ** | + | **65 μL 2 μM chymotrypsin |
| − | * | + | **5 mL H<sub>2</sub>O |
| − | * | + | *20 μL 40% DMSO; prepared such that 1 μL needed per reaction (final concentration = 0.2% in 200 μL) |
| − | *Chembridge ligands (most at 100 mM) | + | *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:''' | '''during the laboratory:''' | ||
| Line 79: | Line 88: | ||
*prepare 5 mM suc-AAFP-pNA in TFE containing 460 mM LiCl | *prepare 5 mM suc-AAFP-pNA in TFE containing 460 mM LiCl | ||
| − | == | + | ==M1D6== |
'''prior to laboratory:''' | '''prior to laboratory:''' | ||
*prepare serial dilutions of reagents (MIX WELL BETWEEN DILUTIONS) | *prepare serial dilutions of reagents (MIX WELL BETWEEN DILUTIONS) | ||
| − | **dye: 1000x → 100x ( | + | **dye: 1000x → 100x (3 μL dye + 27 μL 1X PBS); 100x → 5x (30 μL of 100x dye + 570 μL 1X PBS) |
| − | **DMSO: 100 | + | **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 ( | + | **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 → | + | **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:''' | '''per team:''' | ||
*18 μL 1 mg/mL FKBP12 | *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 | *10 μL 50 μM rapamycin in 1X PBS | ||
| − | *20 μL | + | *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 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== |
'''per team:''' | '''per team:''' | ||
*none | *none | ||
Latest revision as of 14:53, 1 March 2018
M1D1
per team:
- 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
- subculture BL21(DE3) pRSETb_FKBP12 1:10 into 50 mL LB containing 100μg / mL amp and 34 μg / mL cam, 1 per team
per team:
- 25 μL 6x loading dye
- 520 μL 0.1 M IPTG
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
