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Team:Washington/Protocols
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<h2> Media, Plates and Solutions </h2> | <h2> Media, Plates and Solutions </h2> | ||
| - | <h3 | + | <h3>CCMB</h3> |
| - | <h3 | + | |
| + | <h3>LB-Agar </h3> | ||
<p align="left"> | <p align="left"> | ||
10 g tryptone | 10 g tryptone | ||
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</p> | </p> | ||
| - | <h3 | + | <h3>Luria Broth</h3> |
<p align="left"> | <p align="left"> | ||
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 | + | <h3>Super Optimal Broth (S.O.B.)</h3> |
<p align="left"> </p> | <p align="left"> </p> | ||
| - | <h3 | + | <h3>Guanidinium Hydrogen Chloride</h3> |
<p align="left"> </p> | <p align="left"> </p> | ||
| - | <h3 | + | <h3>TB</h3> |
<p align="left"> | <p align="left"> | ||
500mL milliQ filtered water | 500mL milliQ filtered water | ||
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</p> | </p> | ||
<p align="left"> </p> | <p align="left"> </p> | ||
| - | <h3 | + | <h3>YPD</h3> |
<p align="left"> </p> | <p align="left"> </p> | ||
| - | <h3 | + | <h3>Yeast Liquid Cultures</h3> |
<p align="left"> </p> | <p align="left"> </p> | ||
| - | <h3 | + | <h3>PBSF</h3> |
<p align="left"> </p> | <p align="left"> </p> | ||
<h2> Basic Cloning </h2> | <h2> Basic Cloning </h2> | ||
| - | <h3 | + | <h3>Polymerase Chain Reaction (PCR)</h3> |
<p align="left"> </p> | <p align="left"> </p> | ||
| - | <h3 | + | <h3>Restriction Endonuclease Reaction (Digestion)</h3> |
<p align="left"> </p> | <p align="left"> </p> | ||
| - | <h3 | + | <h3>Ligation</h3> |
<p align="left"> </p> | <p align="left"> </p> | ||
<h2> <i> Escherichia coli </i> Protocols (XL1-Blue and XL10-Gold) </h2> | <h2> <i> Escherichia coli </i> Protocols (XL1-Blue and XL10-Gold) </h2> | ||
| - | <h3 | + | <h3>Competent Cell Culturing</h3> |
<p align="left"> </p> | <p align="left"> </p> | ||
| - | <h3 | + | <h3>Competent Cell Transformations</h3> |
<p align="left"> | <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 | ||
</p> | </p> | ||
| - | <h3 | + | <h3> Plating </h3> |
<p align="left"> </p> | <p align="left"> </p> | ||
| - | <h3 | + | <h3> Overnights </h3> |
<p align="left"> </p> | <p align="left"> </p> | ||
| - | <h3 | + | <h3> DNA-Extraction and mini-preps </h3> |
<p align="left"> </p> | <p align="left"> </p> | ||
| - | <h3 | + | <h3> Glycerol Stocks </h3> |
<p align="left"> </p> | <p align="left"> </p> | ||
<h2> <i> Saccharomyces cerevisiae </i> (PYE1 Yeast) </h2> | <h2> <i> Saccharomyces cerevisiae </i> (PYE1 Yeast) </h2> | ||
| - | <h3 | + | <h3> Transformations </h3> |
<p align="left"> </p> | <p align="left"> </p> | ||
| - | <h3 | + | <h3> Overnight Culturing and Passaging </h3> |
<p align="left"> </p> | <p align="left"> </p> | ||
| - | <h3 | + | <h3> Glycerol Stocks </h3> |
<p align="left"> </p> | <p align="left"> </p> | ||
<h2> Flow Cytometry and Fluorescence Activated Cell Sorting </h2> | <h2> Flow Cytometry and Fluorescence Activated Cell Sorting </h2> | ||
| - | <h3 | + | <h3> Dilutions </h3> |
<p align="left"> </p> | <p align="left"> </p> | ||
| - | <h3 | + | <h3> Final Preparations </h3> |
<p align="left"> </p> | <p align="left"> </p> | ||
<h2> Protein Expression </h2> | <h2> Protein Expression </h2> | ||
| - | <h3 | + | <h3> Overnight Cultures </h3> |
<p align="left"> | <p align="left"> | ||
1. In a beveled flask mix: </br> | 1. In a beveled flask mix: </br> | ||
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3. Shake at 37C overnight | 3. Shake at 37C overnight | ||
</p> | </p> | ||
| - | <h3 | + | <h3> Protein Expression </h3> |
<p align="left"> | <p align="left"> | ||
</p> | </p> | ||
| - | <h3 | + | <h3> Protein Extraction and Purification </h3> |
<p align="left"> | <p align="left"> | ||
</p> | </p> | ||
| - | <h3 | + | <h3> Nickel Nitrotriacetic Acid Chromatography </h3> |
<p align="left"> </p> | <p align="left"> </p> | ||
| - | <h3 | + | <h3> Size Exclusion Chromatography (S.E.C.) </h3> |
<p align="left"> </p> | <p align="left"> </p> | ||
<h2> Stability Analysis </h2> | <h2> Stability Analysis </h2> | ||
| - | <h3 | + | <h3>Thermal Melts </h3> |
<p align="left"> </p> | <p align="left"> </p> | ||
| - | <h3 | + | <h3>Guanidinium Hydrogen Chloride Melts </h3> |
<p align="left"> </p> | <p align="left"> </p> | ||
</html> | </html> | ||
Revision as of 21:22, 10 October 2014
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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 swirling 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
