"

From 2014.igem.org

Protocols

Media, Plates and Solutions

CCMB

LB-Agar

10 g tryptone 5 g yeast extract 10 g NaCl 15 g agarose 1 Liter diH2O Autoclave in two 500 ml bottles (liquid cycle, 20 min)

Luria Broth

6.25 g LB mix 250 ml diH2O Autoclave in 500 ml bottle (liquid cycle, 20 min)

Super Optimal Broth (S.O.B.)

Guanidinium Hydrogen Chloride

TB

500mL milliQ filtered water 6g BactoTryptone 12g Yeast Extract 2mL glycerol

YPD

Yeast Liquid Cultures

PBSF

Basic Cloning

Polymerase Chain Reaction (PCR)

Restriction Endonuclease Reaction (Digestion)

Ligation

Escherichia coli Protocols (XL1-Blue and XL10-Gold)

Competent Cell Culturing

Competent Cell Transformations

1. Thaw competent E.coli cells on ice (XL1-Blue for cloning)
2. Add 50 uL of competent cells to sterile 14 mL Falcon culture tubes
3. Add 1 uL of the miniprep to each culture tube
4. Equilibrate the cells on ice for 10 min
5. Heat shock the cells at 42C for 30-45 seconds
6. Immediately place the cells back on ice for 3 min
7. Add 250 uL LB media and shake at 250 rpm and 37C for 30 min
8. Plate 10 ul and 290 ul of the recovered cells onto LB-agar plates supplemented with appropriate antibiotics
9. Invert and incubate at 37C overnight

Plating

Overnights

DNA-Extraction and mini-preps

Glycerol Stocks

Saccharomyces cerevisiae (PYE1 Yeast)

Transformations

Overnight Culturing and Passaging

Glycerol Stocks

Flow Cytometry and Fluorescence Activated Cell Sorting

Dilutions

Final Preparations

Protein Expression

Overnight Cultures

1. In a beveled flask mix: 3mL TB 3uL Kanamycin 2. Add cells by stabbing glycerol stock with p1000 pipette and swirl in solution 3. Shake at 37C overnight

Protein Expression

Protein Extraction and Purification

Nickel Nitrotriacetic Acid Chromatography

Size Exclusion Chromatography (S.E.C.)

Stability Analysis

Thermal Melts

Guanidinium Hydrogen Chloride Melts