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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 7.1 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

LIVE/DEAD® BacLight - Bacterial Viability Kit