Difference between revisions of "20.109(S18): Prep notes for M1"
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Noreen Lyell (Talk | contribs) (→M2D2) |
Noreen Lyell (Talk | contribs) |
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| Line 5: | Line 5: | ||
==M1D1== | ==M1D1== | ||
*restriction enzymes and buffers | *restriction enzymes and buffers | ||
| + | *filtered pipet tips | ||
==M1D2== | ==M1D2== | ||
'''prior to laboratory:''' | '''prior to laboratory:''' | ||
| + | |||
*prepare 1% agarose gels, 1 per 2 teams | *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 | *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:''' |
*X μL 6x loading dye | *X μL 6x loading dye | ||
*Y μL 1 M IPTG | *Y μL 1 M IPTG | ||
| − | == | + | ==M2D3== |
| − | ''' | + | '''per team:''' |
*lysis buffer reagents | *lysis buffer reagents | ||
| Line 28: | Line 30: | ||
*3.5 mL 1X PBS containing 250 mM imidazole | *3.5 mL 1X PBS containing 250 mM imidazole | ||
| − | == | + | ==M2D4== |
| − | ''' | + | '''prior to laboratory:''' |
| + | |||
| + | *boil prep stained and unstained molecular weight standards, if required | ||
| + | *prepare electrophoresis buffer, 1 chamber volume per 2 teams | ||
| + | |||
| + | '''per team:''' | ||
*20 μL Laemmli buffer | *20 μL Laemmli buffer | ||
| − | * | + | *50 mL coomassie stain |
| + | *BSA reagents | ||
| + | **110 μL albumin | ||
| + | **1300 μL 1X PBS | ||
| + | **X μL BCA Reagent A | ||
| + | **Y μL BCA Reagent B | ||
| + | |||
| + | ==M2D5== | ||
| + | '''per team:''' | ||
| + | |||
| + | *18 μL 1 mg/mL FKBP12 | ||
| + | *buffer reagents | ||
| + | **X μL 1 M Tris-HCL pH=8 | ||
| + | **Y μL 100 mM CaCl<sub>2</sub> | ||
| + | **Z μL chymotrypsin | ||
| + | |||
| + | ==M2D6== | ||
| + | '''per team:''' | ||
| + | |||
| + | *18 μL 1 mg/mL FKBP12 | ||
Revision as of 21:42, 31 January 2018
M1D1
- 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:
- X μL 6x loading dye
- Y μL 1 M IPTG
M2D3
per team:
- lysis buffer reagents
- 250 μL slurry
- 7 mL 1X PBS
- Y μL 1 M MgCl2
- 12 μL DNase
- 1.7 mL 1X PBS containing 10 mM imidazole
- 3.5 mL 1X PBS containing 250 mM imidazole
M2D4
prior to laboratory:
- boil prep 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
- 1300 μL 1X PBS
- X μL BCA Reagent A
- Y μL BCA Reagent B
M2D5
per team:
- 18 μL 1 mg/mL FKBP12
- buffer reagents
- X μL 1 M Tris-HCL pH=8
- Y μL 100 mM CaCl2
- Z μL chymotrypsin
M2D6
per team:
- 18 μL 1 mg/mL FKBP12
