Difference between revisions of "20.109(F19): Prep notes for M1"
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Noreen Lyell (Talk | contribs) (Created page with "<div style="padding: 10px; width: 820px; border: 5px solid #A4A4A4;"> {{Template:20.109(F19)}} ==M1D1== '''48 hr preceding laboratory:''' *seed CHO (350K) in T75 flasks, 1 ...") |
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'''48 hr preceding laboratory:''' | '''48 hr preceding laboratory:''' | ||
*seed CHO (350K) in T75 flasks, 1 per team | *seed CHO (350K) in T75 flasks, 1 per team | ||
| − | *[[cell expansion schedule]] for D1, D3, and D4 prep | + | *[[Media:Fa19 M1 cell seeding schedule.xlsx| cell expansion schedule]] for D1, D3, and D4 prep |
'''in tissue culture room (per team):''' | '''in tissue culture room (per team):''' | ||
| Line 22: | Line 22: | ||
'''in tissue culture room (per team):''' | '''in tissue culture room (per team):''' | ||
| − | * | + | *135 μL 12 mM MMS |
| − | * | + | *8 μL 100 mM As |
| − | * | + | *45 mL PBS |
| − | * | + | *40 mL media |
*not aliquoted: EdU | *not aliquoted: EdU | ||
| Line 35: | Line 35: | ||
*seed CHO (350K) in T75 flasks, 1 per team | *seed CHO (350K) in T75 flasks, 1 per team | ||
**teaching faculty will harvest cells just prior to student use and concentrate to 500K / mL | **teaching faculty will harvest cells just prior to student use and concentrate to 500K / mL | ||
| + | |||
| + | '''prior to laboratory''' | ||
| + | *teaching faculty will prepare Click-iT reaction mix | ||
'''in tissue culture room (per team):''' | '''in tissue culture room (per team):''' | ||
| Line 41: | Line 44: | ||
'''in main lab (per team):''' | '''in main lab (per team):''' | ||
*9 mL 1X TBS | *9 mL 1X TBS | ||
| − | *not aliquoted: Triton-X, BSA, anti-γH2AX antibody | + | *not aliquoted: Triton-X, BSA, anti-γH2AX antibody, mounting media, 1X PBS, LMP solution |
''students will prepare following solutions in class:'' | ''students will prepare following solutions in class:'' | ||
| Line 47: | Line 50: | ||
#blocking solution = 2.5 mL 1X TBS containing 1% BSA | #blocking solution = 2.5 mL 1X TBS containing 1% BSA | ||
#1° antibody solution = 2.5 mL blocking solution containing 1:1000 dilution of anti-γH2AX antibody | #1° antibody solution = 2.5 mL blocking solution containing 1:1000 dilution of anti-γH2AX antibody | ||
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==M1D4== | ==M1D4== | ||
| Line 61: | Line 57: | ||
'''prior to laboratory''' | '''prior to laboratory''' | ||
| − | *prepare CometChips, 1 per team | + | *teaching faculty will prepare CometChips, 1 per team |
'''in tissue culture room (per team):''' | '''in tissue culture room (per team):''' | ||
| − | * | + | *none |
| − | + | ||
| − | + | ||
'''in main lab (per team): | '''in main lab (per team): | ||
| − | *not aliquoted: | + | *50 μL 12 mM MMS |
| + | *10 μL 100 mM As | ||
| + | *12 mL media | ||
| + | |||
| + | *not aliquoted: 1X PBS, LMP solution, alkaline lysis buffer | ||
==M1D5== | ==M1D5== | ||
| + | |||
'''in tissue culture (per team):''' | '''in tissue culture (per team):''' | ||
| − | * | + | *none |
| − | + | ||
| − | + | ||
'''in main lab (per team): | '''in main lab (per team): | ||
| − | * | + | *not aliquoted: unwinding / electrophoresis buffer, neutralization buffer, SYBR gold |
==M1D6== | ==M1D6== | ||
| Line 85: | Line 82: | ||
'''in main lab (per team):''' | '''in main lab (per team):''' | ||
| − | * | + | *none |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
==M1D7== | ==M1D7== | ||
Latest revision as of 17:48, 4 September 2019
M1D1
48 hr preceding laboratory:
- seed CHO (350K) in T75 flasks, 1 per team
- cell expansion schedule for D1, D3, and D4 prep
in tissue culture room (per team):
- 1 fresh 12-well plate
- 5.5 mL PBS
- 2.5 mL trypsin
- 35 mL media
- not aliquoted: trypan blue
in main lab (per team):
- none
M1D2
in tissue culture room (per team):
- 135 μL 12 mM MMS
- 8 μL 100 mM As
- 45 mL PBS
- 40 mL media
- not aliquoted: EdU
in main lab (per team):
- not aliquoted: NMP stock, 1X PBS, gel bond film (pre-cut), markers, rulers
M1D3
48 hr preceding laboratory:
- seed CHO (350K) in T75 flasks, 1 per team
- teaching faculty will harvest cells just prior to student use and concentrate to 500K / mL
prior to laboratory
- teaching faculty will prepare Click-iT reaction mix
in tissue culture room (per team):
- none
in main lab (per team):
- 9 mL 1X TBS
- not aliquoted: Triton-X, BSA, anti-γH2AX antibody, mounting media, 1X PBS, LMP solution
students will prepare following solutions in class:
- Triton-X solution = 2.5 mL 1X TBS containing 0.2% Triton-X
- blocking solution = 2.5 mL 1X TBS containing 1% BSA
- 1° antibody solution = 2.5 mL blocking solution containing 1:1000 dilution of anti-γH2AX antibody
M1D4
48 hr preceding laboratory:
- seed CHO (350K) in T75 flasks, 1 per team
- teaching faculty will harvest cells just prior to student use and concentrate to 500K / mL
prior to laboratory
- teaching faculty will prepare CometChips, 1 per team
in tissue culture room (per team):
- none
in main lab (per team):
- 50 μL 12 mM MMS
- 10 μL 100 mM As
- 12 mL media
- not aliquoted: 1X PBS, LMP solution, alkaline lysis buffer
M1D5
in tissue culture (per team):
- none
in main lab (per team):
- not aliquoted: unwinding / electrophoresis buffer, neutralization buffer, SYBR gold
M1D6
in tissue culture room (per team):
- none
in main lab (per team):
- none
M1D7
in tissue culture room (per team):
- none
in main lab (per team):
- none
