Team:Northwestern/Protocol
From 2010.igem.org
(Difference between revisions)
(→Rhodamine-Conjugated Chitin Probe) |
(→Rhodamine-Conjugated Chitin Probe) |
||
Line 76: | Line 76: | ||
=='''Cell Staining'''== | =='''Cell Staining'''== | ||
===[[Rhodamine-Conjugated Chitin Probe]]=== | ===[[Rhodamine-Conjugated Chitin Probe]]=== | ||
- | Methanol Fixation | + | ==Methanol Fixation== |
# Rinse slide with ethanol and flame | # Rinse slide with ethanol and flame | ||
# Rinse slide with PBS and dry with KimWipe | # Rinse slide with PBS and dry with KimWipe | ||
Line 82: | Line 82: | ||
# Submerge in -20C absolute methanol for 5-10 min | # Submerge in -20C absolute methanol for 5-10 min | ||
# Wash 3 times with 1X TBS (submerge in 1X TBS for 5 minutes per wash) | # Wash 3 times with 1X TBS (submerge in 1X TBS for 5 minutes per wash) | ||
- | * '''Do not let cells dry for the rest of the procedure''' | + | ** '''Do not let cells dry for the rest of the procedure''' |
- | Staining | + | ==Staining== |
# Make 1:500 dilution of chitin probe in TBS (50ul of probe is used per slide, [49.9ul of 1X TBS]:[0.1ul of chitin probe] --> 1 slide) | # Make 1:500 dilution of chitin probe in TBS (50ul of probe is used per slide, [49.9ul of 1X TBS]:[0.1ul of chitin probe] --> 1 slide) | ||
# Apply 50ul to the slide containing the sample | # Apply 50ul to the slide containing the sample | ||
# Allow stain to incubate overnight at RT in a dark room | # Allow stain to incubate overnight at RT in a dark room | ||
- | * ''To prevent stain from evaporating overnight'': Use a large glass petri dish. Soak a paper towel with PBS and place it at the bottom of the petri dish. Use 2 toothpicks to support the slide above the paper towel. This prevents any wicking action from occurring and pulling the stain off the slide. | + | ** ''To prevent stain from evaporating overnight'': Use a large glass petri dish. Soak a paper towel with PBS and place it at the bottom of the petri dish. Use 2 toothpicks to support the slide above the paper towel. This prevents any wicking action from occurring and pulling the stain off the slide. |
# Wash 3 times with 1X TBS (submerge in 1X TBS for 5 minutes per wash) | # Wash 3 times with 1X TBS (submerge in 1X TBS for 5 minutes per wash) | ||
===[[LIVE/DEAD® BacLight - Bacterial Viability Kit]]=== | ===[[LIVE/DEAD® BacLight - Bacterial Viability Kit]]=== |
Revision as of 19:27, 14 August 2010
Home | Brainstorm | Team | Acknowledgements | Project | Side Project | Parts | Notebook | Calendar | Protocol | Safety | Links | References | Media | Contact |
---|
Contents |
DNA
Prepping DNA from the kit plates
Quikchange (from primers to colonies!)
Kit to Stock Plasmid
Ethanol Precipitation
New Part Design(PCR)
Bacterial Work
Transformation
O/N Culture
Preparation of Competent Cells
LB Media
Preparing Plates
Glycerol Stocks
Assembly
Restriction Enzyme Digests
Plasmid Construction
3A Assembly
Ligations
Mini Prep
Microscopy
Confocal Microscopy
Fluorescence Microscopy
Reagents
Reagents
Cell Staining
Rhodamine-Conjugated Chitin Probe
Methanol Fixation
- Rinse slide with ethanol and flame
- Rinse slide with PBS and dry with KimWipe
- Apply cell sample to microscope slide and let air dry
- Submerge in -20C absolute methanol for 5-10 min
- Wash 3 times with 1X TBS (submerge in 1X TBS for 5 minutes per wash)
- Do not let cells dry for the rest of the procedure
Staining
- Make 1:500 dilution of chitin probe in TBS (50ul of probe is used per slide, [49.9ul of 1X TBS]:[0.1ul of chitin probe] --> 1 slide)
- Apply 50ul to the slide containing the sample
- Allow stain to incubate overnight at RT in a dark room
- To prevent stain from evaporating overnight: Use a large glass petri dish. Soak a paper towel with PBS and place it at the bottom of the petri dish. Use 2 toothpicks to support the slide above the paper towel. This prevents any wicking action from occurring and pulling the stain off the slide.
- Wash 3 times with 1X TBS (submerge in 1X TBS for 5 minutes per wash)