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Team:Washington/Protocols
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<h1> Protocols </h1> | <h1> Protocols </h1> | ||
<h2> Media, Plates and Solutions </h2> | <h2> Media, Plates and Solutions </h2> | ||
| - | <h3>CCMB</h3> | + | <h3> CCMB </h3> |
| - | <h3>LB-Agar </h3> | + | <h3> LB-Agar </h3> |
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10 g tryptone | 10 g tryptone | ||
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| - | <h3>Luria Broth</h3> | + | <h3> Luria Broth </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) | ||
</p> | </p> | ||
| - | <h3>Super Optimal Broth (S.O.B.)</h3> | + | <h3> Super Optimal Broth (S.O.B.) </h3> |
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| - | <h3>Guanidinium Hydrogen Chloride</h3> | + | <h3> Guanidinium Hydrogen Chloride </h3> |
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| - | + | <h3> YPD </h3> | |
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| - | + | <h3> Yeast Liquid Cultures </h3> | |
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| - | + | <h3> PBSF </h3> | |
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| - | <h3>YPD</h3> | + | |
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| - | <h3>Yeast Liquid Cultures</h3> | + | |
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| - | <h3>PBSF</h3 | + | |
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<h2> Basic Cloning </h2> | <h2> Basic Cloning </h2> | ||
| - | <h3>Polymerase Chain | + | <h3> Polymerase Chain Reactions </h3> |
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| - | <h3>Restriction Endonuclease Reaction (Digestion)</h3> | + | <h3> Restriction Endonuclease Reaction (Digestion) </h3> |
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| - | <h3>Ligation</h3 | + | <h3> Ligation </h3> |
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| - | <h2> <i> Escherichia coli | + | <h2> <i> Escherichia coli Protocols (XL1-Blue and XL10-Gold) </i> </h2> |
| - | <h3>Competent Cell Culturing</h3> | + | <h3> Competent Cell Culturing </h3> |
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| - | <h3>Competent Cell Transformations</h3> | + | <h3> Competent Cell Transformations </h3> |
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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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<h3> Plating </h3> | <h3> Plating </h3> | ||
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<h3> Overnights </h3> | <h3> Overnights </h3> | ||
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<h3> DNA-Extraction and mini-preps </h3> | <h3> DNA-Extraction and mini-preps </h3> | ||
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<h3> Glycerol Stocks </h3> | <h3> Glycerol Stocks </h3> | ||
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| - | <h2> <i> Saccharomyces cerevisiae </i> | + | <h2> <i> Saccharomyces cerevisiae (PYE1 Yeast)</i> </h2> |
<h3> Transformations </h3> | <h3> Transformations </h3> | ||
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| - | <h3> | + | <h3> OVernight Culturing and Passaging </h3> |
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<h3> Glycerol Stocks </h3> | <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> | ||
<h3> Dilutions </h3> | <h3> Dilutions </h3> | ||
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<h3> Final Preparations </h3> | <h3> Final Preparations </h3> | ||
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<h2> Protein Expression </h2> | <h2> Protein Expression </h2> | ||
<h3> Overnight Cultures </h3> | <h3> Overnight Cultures </h3> | ||
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| - | + | <h3> Protein Extraction and Purification </h3> | |
| - | <h3> Protein Extraction and Purification </h3 | + | |
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<h3> Nickel Nitrotriacetic Acid Chromatography </h3> | <h3> Nickel Nitrotriacetic Acid Chromatography </h3> | ||
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<h3> Size Exclusion Chromatography (S.E.C.) </h3> | <h3> Size Exclusion Chromatography (S.E.C.) </h3> | ||
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<h2> Stability Analysis </h2> | <h2> Stability Analysis </h2> | ||
| - | <h3>Thermal Melts </h3> | + | <h3> Thermal Melts </h3> |
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| - | <h3>Guanidinium Hydrogen Chloride Melts </h3 | + | <h3> Guanidinium Hydrogen Chloride Melts</h3> |
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Revision as of 00:47, 12 October 2014
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Contents |
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 Reactions
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
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