Team:Northwestern/Protocol

DNA

Prepping DNA from the kit plates

1. With a pipette tip, punch a hole through the foil cover into the corresponding well to the Biobrick™-standard part that you want. Make sure you have properly oriented the plate. We recommend that you do not remove the foil cover, as it could lead to cross contamination between the wells.
2. Add 10uL of diH2O (deionized water)
3. Pipette 1 or 2uL of the resuspended DNA Transformation into your desired competent cells, plate bacteria with the appropriate antibiotic* and grow overnight.
4. Pick a single colony and inoculate broth (again, with the correct antibiotic) and grow for 18 hours.
5. Use the resulting culture to miniprep the DNA AND make your own glycerol stock (for further instruction on making a glycerol see this page). We recommend using the miniprepped DNA to run QC tests, such as restriction digests and sequencing.

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