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Contents 1 DNA 1.1 Prepping DNA from the kit plates 1.2 Quikchange (from primers to colonies!) 1.3 Kit to Stock Plasmid 1.4 Ethanol Precipitation 1.5 New Part Design(PCR) 2 Bacterial Work 2.1 Transformation 2.2 O/N Culture 2.3 Preparation of Competent Cells 2.4 LB Media 2.5 Preparing Plates 2.6 Glycerol Stocks 3 Assembly 3.1 Restriction Enzyme Digests 3.2 Plasmid Construction 3.3 3A Assembly 3.4 Ligations 3.5 Mini Prep 4 Microscopy 4.1 Confocal Microscopy 4.2 Fluorescence Microscopy 5 Reagents 5.1 Reagents 6 Cell Staining 6.1 Rhodamine-Conjugated Chitin Probe 7 Methanol Fixation 8 Staining 8.1 LIVE/DEAD® BacLight - Bacterial Viability Kit

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

1. Rinse slide with ethanol and flame
2. Rinse slide with PBS and dry with KimWipe
3. Apply cell sample to microscope slide and let air dry
4. Submerge in -20C absolute methanol for 5-10 min
5. 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

1. 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)
2. Apply 50ul to the slide containing the sample
3. 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.

1. Wash 3 times with 1X TBS (submerge in 1X TBS for 5 minutes per wash)

LIVE/DEAD® BacLight - Bacterial Viability Kit