January 14
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Introduction the laboratory-Tour of the lab,
set up lab groups, check the equipment list
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January 16
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Make your first medium
; check for contamination
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January 21
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Check new medium for contamination;
Set up Basic Cell Culture #1 Experiment
.
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22
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Conduct 24 hour count
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23
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Conduct 48 hour count
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24
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Conduct 72 hour count
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January 28
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Redo Basic Cell
Culture #1 Experiment
; Freeze cells for storage
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29
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Conduct 24 hour count
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30
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Conduct 48 hour count; Unfreeze test cells
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31
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Conduct 72 hour count
|
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February 4
|
Discuss Basic Cell Culture #1 Experiment; Set
up Basic Cell Culture #3 Experiment
; Freeze down your own cells for storage
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6
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Conduct 24 hour count
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7
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Conduct 48 hour count
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8
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Conduct 72 hour count
|
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February 11
|
LABORATORY MEETING: Discussion of ALL
growth experiments; Discuss setup of Serum-Free Experiment
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February 13
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Set up Serum-Free
Growth Experiment
|
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February 15
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Conduct 48 hour count
|
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February 17
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Conduct 96 hour count
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18
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Set up Soft agar Experiment
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19
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Conduct 144 hour count
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20
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Stain and count plates from Serum-Free Experiment
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February 25
|
Set up for Karyology
and Mycoplasma
Experiments; Count Soft Agar Experiment plates
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February 27
|
Do Karyology Experiment
(i.e.obtain and stain chromosome spreads and observe and count) or store
them for the next day
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28
|
Stain and count spreads
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March 4
|
TOWN MEETING DAY - NO CLASS OR LAB (i.e.
unless you have any finishing up to do)
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March 6
|
LAB MEETING: Discuss Serum-Free,
Soft Agar & Karyology Data; Set up Mycoplasma
Exp't.
- REMEMBER, NO ANTIBIOTICS;
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March 11
|
Stain for mycoplasma and view in fluorescent
scope
|
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March 13
|
LAB MEETING: Discuss Mycoplasma data
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March 17-21
|
****************SPRING BREAK -- ENJOY*******************
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March 25
|
Obtain T25 flasks of C2C12 cells to be used
in transfection experiments, make observations;
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March 27
|
Harvest, count and reseed cells for stock; Switch
C2C12 media to induce differentiation - DAY 1
|
28
|
Observe C2C12 cultures - DAY 2 and replace
media
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29
|
Add 10 ml fresh media to T75 stock flasks; Observe
C2C12 cultures - DAY 3 and replace media
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30
|
Observe C2C12 cultures - DAY 4 and replace
media
|
31
|
Harvest, count and seed cells in chamberslides
for transfection tomorrow--REMEMBER TO HARVEST IN ANTIBIOTIC-FREE MEDIA;
make new stock flasks; Observe C2C12 cultures - DAY 5
and replace media
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April 1
|
Transfect cells with myoD or pEGFP-N2 using
LipofectAMINE PLUS; Observe C2C12 cultures - DAY 6 and replace media
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2
|
Observe C2C12 cultures - DAY 7 and replace
media, Change media on chamberslides
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3
|
Fix cells and perform indirect immunofluorescence
using anti-myosin and anti-myoD and store slides at 4o C; Split
stocks; Observe C2C12 cultures - DAY 8
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April 5
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Add 10 ml fresh media to T75 stock flasks
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April 7
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Harvest, count and seed cells in T25's for transfection
tomorrow; REMEMBER TO HARVEST IN ANTIBIOTIC-FREE MEDIA
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8
|
Transfect cells with myoD using LipofectAMINE
PLUS; View indirect immunofluorescence slides
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9
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Change media in T25's
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10
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Harvest cells and extract proteins and freeze
extracts; View indirect immunofluorescence slides
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April 15
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Make 6% and 15% SDS-PAGE resolving gels and
buffers; View indirect immunofluorescence slides
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April 17
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Pour 4% SDS-PAGE stacking gels; Run protein
extracts on SDS-PAGE gels (2); Transfer proteins from gels to nylon membrane;
Stain transferred gel with Coomassie and destain
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April 22
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Probe Westerns with anti-myoD and anti-myosin
and detect with the ECL kit; Expose to film; Dry Coomassie-stained gels
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April 24
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LAB MEETING: Present data on indirect
immunofluorescence and Western results
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April 29
|
Class Project Presentations - one in lecture
and two in lab. - LAST DAY OF CLASSES
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