Protocols

LB Media

950 mL H₂0

10 g Tryptone

10 g NaCl

5 g Yeast Extract

1 L Total

Add 15 g Agar, if being poured into plates.

Autoclave, when cool add antibiotics if desired.

Antibiotic Stock Solutions

Chloramphenicol

Working Concentration 12.5 μg/ml

Stock solutions can be made at 35 mg/ml in ethanol and kept at -20º C

Kanamycin

Filter sterilize for kanamycin

Working Concentration:

25 μg/mL for low-copy plasmids

35 µg/mL for high-copy plasmids

Stock solution is 35 mg/ml in water and kept at -20º C

Spectinomycin

Filter sterilize

Working Concentration 50 μg/mL

Stock solution is 100 mg/mL in water and kept at -20º C

Heat Shock Transformation

1. Preheat water bath to 42º C.
2. Thaw competent cells on ice for 10 minutes.
3. Use 50 µL competent cells, add transforming DNA [up to 25 ng per 50 µL of cells, volume not exceeding 2.5 µL (5%)]. For control add 1 µL of control DNA (PUC19 carb resistance). Swirl to mix, store on ice 5 minutes.
4. Heat shock in 42º C water bath for 45 seconds.
5. Cool cells in ice bath for a few minutes.
6. Add 800 µL LB to each tube, set in shaker at 37º C for 45 minutes.
7. Transfer 200 µL of culture per plate (containing the appropriate antibiotic).
8. Spread using glass beads.
9. Invert plates and incubate overnight (12-16 hrs) at 37º C.< /li>

Making Chemically Competent Cells

Double Restriction (Fast) Digest

Ligations (Standard Assembly)

Gel Electrophoresis

Gel Extraction and Purification

PCR Amplification for Golden Gate Assembly

Golden Gate Assembly

DNA Extraction (Minipreps)

Making Glycerol Stocks

Sequencing Prep

Tecan Testing Protocol

Site-Directed Mutagenesis PCR

Electroporation Transformation