Team:Washington/Protocols

From 2014.igem.org

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<h1> Protocols </h1>
<h1> Protocols </h1>
<h2> Media, Plates and Solutions </h2>
<h2> Media, Plates and Solutions </h2>
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<h3> CCMB </h3>
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<h3><p align="left"> CCMB </p></h3>
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<h3><p align="left"> LB-Agar </p></h3>
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<h3> LB-Agar </h3>
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10 g tryptone
10 g tryptone
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<h3> Luria Broth </h3>
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<h3><p align="left"> Luria Broth </p></h3>
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6.25 g LB mix  
6.25 g LB mix  
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Autoclave in 500 ml bottle (liquid cycle, 20 min)
Autoclave in 500 ml bottle (liquid cycle, 20 min)
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<h3> Super Optimal Broth (S.O.B.) </h3>
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<h3><p align="left"> Super Optimal Broth (S.O.B.) </p></h3>
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<p align="left"> </p>
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<h3> Guanidinium Hydrogen Chloride </h3>
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<h3><p align="left"> Guanidinium Hydrogen Chloride </p> </h3>
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<p align="left"> </p>
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<h3> YPD </h3>
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<h3><p align="left"> YPD </p></h3>
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<p align="left"> </p>
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<h3> Yeast Liquid Cultures </h3>
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<h3><p align="left"> Yeast Liquid Cultures </p></h3>
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<p align="left"> </p>
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<h3> PBSF </h3>
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<h3><p align="left"> PBSF </p></h3>
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<p align="left"> </p>
<h2> Basic Cloning </h2>
<h2> Basic Cloning </h2>
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<h3> Polymerase Chain Reactions </h3>
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<h3><p align="left"> Polymerase Chain Reaction (PCR) </p></h3>
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<p align="left"> </p>
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<h3> Restriction Endonuclease Reaction (Digestion) </h3>
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<h3><p align="left"> Restriction Endonuclease Reaction (Digestion) </p></h3>
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<p align="left"> </p>
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<h3> Ligation </h3>
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<h3><p align="left"> Ligation </p></h3>
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<p align="left"> </p>
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<h2> <i> Escherichia coli Protocols (XL1-Blue and XL10-Gold) </i> </h2>
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<h2> <i> Escherichia coli </i> Protocols (XL1-Blue and XL10-Gold) </h2>
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<h3> Competent Cell Culturing </h3>
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<h3><p align="left"> Competent Cell Culturing </p></h3>
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<p align="left"> </p>
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<h3> Competent Cell Transformations </h3>
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<h3><p align="left"> Competent Cell Transformations </p></h3>
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<p align="left">
1.  Thaw competent E.coli cells on ice (XL1-Blue for cloning) <br>
1.  Thaw competent E.coli cells on ice (XL1-Blue for cloning) <br>
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9.  Invert and incubate at 37C overnight
9.  Invert and incubate at 37C overnight
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<h3> Plating </h3>
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<h3><p align="left"> Plating </p></h3>
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<p align="left"> </p>
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<h3> Overnights </h3>
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<h3><p align="left"> Overnights </p></h3>
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<p align="left"> </p>
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<h3><p align="left"> DNA-Extraction and mini-preps </p></h3>
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<p align="left"> </p>
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<h3><p align="left"> Glycerol Stocks </p></h3>
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<p align="left"> </p>
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<h2> <i> Saccharomyces cerevisiae </i> (PYE1 Yeast) </h2>
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<h3> DNA-Extraction and mini-preps </h3>
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<h3><p align="left"> Transformations </p></h3>
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<p align="left"> </p>
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<h3> Glycerol Stocks </h3>
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<h3><p align="left"> OVernight Culturing and Passaging </p></h3>
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<p align="left"> </p>
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<h2> <i> Saccharomyces cerevisiae (PYE1 Yeast)</i> </h2>
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<h3><p align="left"> Glycerol Stocks </p></h3>
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<p align="left"> </p>
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<h3> Transformations </h3>
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<h3> OVernight Culturing and Passaging </h3>
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<h3> Glycerol Stocks </h3>
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<h2> Flow Cytometry and Fluorescence Activated Cell Sorting </h2>
<h2> Flow Cytometry and Fluorescence Activated Cell Sorting </h2>
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<h3> Dilutions </h3>
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<h3><p align="left"> Dilutions </p></h3>
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<p align="left"> </p>
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<h3> Final Preparations </h3>
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<h3><p align="left"> Final Preparations </p></h3>
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<p align="left"> </p>
<h2> Protein Expression </h2>
<h2> Protein Expression </h2>
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<h3> Overnight Cultures </h3>
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<h3><p align="left"> Overnight Cultures </p></h3>
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<p align="left"> </p>
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<h3> Protein Extraction and Purification </h3>
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<h3><p align="left"> Protein Extraction and Purification </p></h3>
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<p align="left"> </p>
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<h3> Nickel Nitrotriacetic Acid Chromatography </h3>
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<h3><p align="left"> Nickel Nitrotriacetic Acid Chromatography </p></h3>
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<p align="left"> </p>
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<h3> Size Exclusion Chromatography (S.E.C.) </h3>
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<h3><p align="left"> Size Exclusion Chromatography (S.E.C.) </p></h3>
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<p align="left"> </p>
<h2> Stability Analysis </h2>
<h2> Stability Analysis </h2>
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<h3> Thermal Melts </h3>
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<h3><p align="left"> Thermal Melts </p></h3>
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<p align="left"> </p>
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<h3> Guanidinium Hydrogen Chloride Melts</h3>
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<h3><p align="left"> Guanidinium Hydrogen Chloride Melts </p></h3>
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<p align="left"> </p>
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Revision as of 03:45, 10 October 2014

UW Homepage Official iGEM website

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

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

Protein Extraction and Purification

Nickel Nitrotriacetic Acid Chromatography

Size Exclusion Chromatography (S.E.C.)

Stability Analysis

Thermal Melts

Guanidinium Hydrogen Chloride Melts

Retrieved from "http://2014.igem.org/Team:Washington/Protocols"