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<br> | <br> | ||
<h5>*Note: Pipettes are needed.</h5> | <h5>*Note: Pipettes are needed.</h5> | ||
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<h3>Making the Agarose Gel</h3> | <h3>Making the Agarose Gel</h3> | ||
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<h5>• Microwave</h5> | <h5>• Microwave</h5> | ||
<h5>• Gel mold</h5> | <h5>• Gel mold</h5> | ||
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<h4>Directions</h4> | <h4>Directions</h4> | ||
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<h5>7. Cool until solidified.</h5> | <h5>7. Cool until solidified.</h5> | ||
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<h3>Gel Electrophoresis</h3> | <h3>Gel Electrophoresis</h3> | ||
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<h5>• Purple loading dye</h5> | <h5>• Purple loading dye</h5> | ||
<h5>• Gel box</h5> | <h5>• Gel box</h5> | ||
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<h4>Directions</h4> | <h4>Directions</h4> | ||
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<h5>8. Results can now be analyzed.</h5> | <h5>8. Results can now be analyzed.</h5> | ||
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<h5>• 25μL Q5 2x High-Fidelity MasterMix</h5> | <h5>• 25μL Q5 2x High-Fidelity MasterMix</h5> | ||
<h5>• 19.9μL NFW (nuclease free water)</h5> | <h5>• 19.9μL NFW (nuclease free water)</h5> | ||
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<h4>Materials: *</h4> | <h4>Materials: *</h4> | ||
Line 67: | Line 77: | ||
<h5>• Thermocycler</h5> | <h5>• Thermocycler</h5> | ||
<h5>• Mini microfuge PCR tube(s)</h5> | <h5>• Mini microfuge PCR tube(s)</h5> | ||
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<h4>Directions:</h4> | <h4>Directions:</h4> | ||
Line 81: | Line 93: | ||
<h5>Step 8: End</h5> | <h5>Step 8: End</h5> | ||
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<h3>PCR Purification of DNA</h3> | <h3>PCR Purification of DNA</h3> | ||
Line 91: | Line 104: | ||
<h5>• EZ-10 column(s)</h5> | <h5>• EZ-10 column(s)</h5> | ||
<h5>• 1.5mL microfuge tube(s)</h5> | <h5>• 1.5mL microfuge tube(s)</h5> | ||
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<h4>Directions: (for each PCR reaction)</h4> | <h4>Directions: (for each PCR reaction)</h4> | ||
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<h5>8. Store at -20 degrees Celsius, or nanodrop for concentration and for dilutions (see Parts Dilutions Protocol).</h5> | <h5>8. Store at -20 degrees Celsius, or nanodrop for concentration and for dilutions (see Parts Dilutions Protocol).</h5> | ||
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<h3>Parts Dilutions</h3> | <h3>Parts Dilutions</h3> | ||
Line 107: | Line 123: | ||
<h5>• PCR Purified DNA Part Reaction (nanodropped with concentration)</h5> | <h5>• PCR Purified DNA Part Reaction (nanodropped with concentration)</h5> | ||
<h5>• 1.5 mL microfuge tube</h5> | <h5>• 1.5 mL microfuge tube</h5> | ||
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<h4>Directions</h4> | <h4>Directions</h4> | ||
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<h5>8. Store at -20 degrees Celsius.</h5> | <h5>8. Store at -20 degrees Celsius.</h5> | ||
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<h3>Golden Gate Assembly Assembly Protocol</h3> | <h3>Golden Gate Assembly Assembly Protocol</h3> | ||
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<h5>• 2μL: T4 Ligase (2M cohesive units)</h5> | <h5>• 2μL: T4 Ligase (2M cohesive units)</h5> | ||
<h5>• 5.35μL NFW</h5> | <h5>• 5.35μL NFW</h5> | ||
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<h4>Materials: *</h4> | <h4>Materials: *</h4> | ||
Line 137: | Line 158: | ||
<h5>• Thermocycler</h5> | <h5>• Thermocycler</h5> | ||
<h5>•Mini microfuge tube(s)</h5> | <h5>•Mini microfuge tube(s)</h5> | ||
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<h4>Directions</h4> | <h4>Directions</h4> | ||
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<h5>Step 7: End</h5> | <h5>Step 7: End</h5> | ||
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<h3>PCR Check for Golden Gate Plasmids</h3> | <h3>PCR Check for Golden Gate Plasmids</h3> | ||
Line 160: | Line 184: | ||
<h5>• 5μL 2x MasterMix</h5> | <h5>• 5μL 2x MasterMix</h5> | ||
<h5>• 3.9μL NFW</h5> | <h5>• 3.9μL NFW</h5> | ||
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<h4>Materials *</h4> | <h4>Materials *</h4> | ||
Line 165: | Line 191: | ||
<h5>• Thermocycler</h5> | <h5>• Thermocycler</h5> | ||
<h5>• Mini microfuge tube(s)</h5> | <h5>• Mini microfuge tube(s)</h5> | ||
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<h4>Directions</h4> | <h4>Directions</h4> | ||
Line 179: | Line 207: | ||
<h5>Step 8: End</h5> | <h5>Step 8: End</h5> | ||
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<h3>Making LB Media (& Autoclaving)</h3> | <h3>Making LB Media (& Autoclaving)</h3> | ||
Line 184: | Line 213: | ||
<h5>• LB powder</h5> | <h5>• LB powder</h5> | ||
<h5>• Distilled water</h5> | <h5>• Distilled water</h5> | ||
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<h4>Materials</h4> | <h4>Materials</h4> | ||
Line 189: | Line 220: | ||
<h5>• Glass bottle</h5> | <h5>• Glass bottle</h5> | ||
<h5>• Autoclave</h5> | <h5>• Autoclave</h5> | ||
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<h4>Directions</h4> | <h4>Directions</h4> | ||
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<h5>3. Add 250mL distilled water to bottle.</h5> | <h5>3. Add 250mL distilled water to bottle.</h5> | ||
<h5>4. Autoclave for ~30 min.</h5> | <h5>4. Autoclave for ~30 min.</h5> | ||
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<h4>To Autoclave</h4> | <h4>To Autoclave</h4> | ||
Line 210: | Line 245: | ||
<h5>12. Open manual valves and release steam.</h5> | <h5>12. Open manual valves and release steam.</h5> | ||
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<h3>Making LB Media w/ Antibiotic Resistance for Plates</h3> | <h3>Making LB Media w/ Antibiotic Resistance for Plates</h3> | ||
Line 217: | Line 253: | ||
<h5>• 4.5g Agar Powder</h5> | <h5>• 4.5g Agar Powder</h5> | ||
<h5>• Antibiotic (usually use Kanamycin - 1μL per 1mL H2O)</h5> | <h5>• Antibiotic (usually use Kanamycin - 1μL per 1mL H2O)</h5> | ||
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<h4>Materials: *</h4> | <h4>Materials: *</h4> | ||
Line 222: | Line 260: | ||
<h5>• Scale</h5> | <h5>• Scale</h5> | ||
<h5>• Glass bottle</h5> | <h5>• Glass bottle</h5> | ||
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<h4>Directions</h4> | <h4>Directions</h4> | ||
Line 231: | Line 271: | ||
<h5>6. Open the lid to each plate carefully and pour plate near the flame.</h5> | <h5>6. Open the lid to each plate carefully and pour plate near the flame.</h5> | ||
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<h3>Bacterial Transformation of Plasmids (& Growing Liquid Cultures)</h3> | <h3>Bacterial Transformation of Plasmids (& Growing Liquid Cultures)</h3> | ||
Line 245: | Line 286: | ||
<h5>• Petri Plates with LB agar and antibiotic</h5> | <h5>• Petri Plates with LB agar and antibiotic</h5> | ||
<h5>• Sterile Spreader or sterile glass beads</h5> | <h5>• Sterile Spreader or sterile glass beads</h5> | ||
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<h4>Directions</h4> | <h4>Directions</h4> | ||
Line 258: | Line 301: | ||
<h5>10. Pipette each transformation on petri plates (labelled!).</h5> | <h5>10. Pipette each transformation on petri plates (labelled!).</h5> | ||
<h5>11. Incubate transformations overnight (14-18 hours) at 37°C.</h5> | <h5>11. Incubate transformations overnight (14-18 hours) at 37°C.</h5> | ||
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<h4>The Next Day</h4> | <h4>The Next Day</h4> | ||
Line 266: | Line 311: | ||
<h5>5. Incubate gridded plate and the liquid cultures at 37°C overnight. Take out the next morning and store in the refrigerator (4°C).</h5> | <h5>5. Incubate gridded plate and the liquid cultures at 37°C overnight. Take out the next morning and store in the refrigerator (4°C).</h5> | ||
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<h3>Plate Reading (for Fluorescence, Absorbance, Induction, etc.)</h3> | <h3>Plate Reading (for Fluorescence, Absorbance, Induction, etc.)</h3> | ||
Line 273: | Line 319: | ||
<h5>• Plate Reader (we use VICTOR X3)</h5> | <h5>• Plate Reader (we use VICTOR X3)</h5> | ||
<h5>• LB Media</h5> | <h5>• LB Media</h5> | ||
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<h4>Directions</h4> | <h4>Directions</h4> | ||
Line 290: | Line 338: | ||
<h5>14. To analyze data using Python program—file must be in a csv format. (If you would like the code for analyzing this type of data, please contact us!)</h5> | <h5>14. To analyze data using Python program—file must be in a csv format. (If you would like the code for analyzing this type of data, please contact us!)</h5> | ||
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<h3>Purification of Plasmid DNA</h3> | <h3>Purification of Plasmid DNA</h3> | ||
Line 295: | Line 344: | ||
<h5>• Liquid cultures</h5> | <h5>• Liquid cultures</h5> | ||
<h5>• Miniprep DNA kit (we used BioBasic kit)</h5> | <h5>• Miniprep DNA kit (we used BioBasic kit)</h5> | ||
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<h4>Directions</h4> | <h4>Directions</h4> | ||
Line 310: | Line 361: | ||
<h5>11. Transfer the column to a clean 1.5mL microfuge tube. Add 50μL of Elution Buffer into the center part of the column and incubate at room temperature for 2 minutes. Centrifuge at 10,000rpm for 2 minutes. </h5> | <h5>11. Transfer the column to a clean 1.5mL microfuge tube. Add 50μL of Elution Buffer into the center part of the column and incubate at room temperature for 2 minutes. Centrifuge at 10,000rpm for 2 minutes. </h5> | ||
<h5>12. Store purified DNA at -20°C.</h5> | <h5>12. Store purified DNA at -20°C.</h5> | ||
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<h4>For centrifuging culture to pellets:</h4> | <h4>For centrifuging culture to pellets:</h4> | ||
Line 318: | Line 371: | ||
<h5>5. Pour off rest.</h5> | <h5>5. Pour off rest.</h5> | ||
<h5>6. Freeze.</h5> | <h5>6. Freeze.</h5> | ||
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+ | <br> | ||
<h3>Plate Reading (for Fluorescence using TX-TL for GG105-108 w/ dcas9 expression plasmids)</h3 | <h3>Plate Reading (for Fluorescence using TX-TL for GG105-108 w/ dcas9 expression plasmids)</h3 | ||
Line 329: | Line 384: | ||
<h5>• GG reaction plasmids w/ clamp binding sites (in our case, GG105-108)</h5> | <h5>• GG reaction plasmids w/ clamp binding sites (in our case, GG105-108)</h5> | ||
<h5>• 384-well plate</h5> | <h5>• 384-well plate</h5> | ||
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<h4>Directions: </h4> | <h4>Directions: </h4> | ||
Line 346: | Line 403: | ||
<h5> For more information on TX-TL, see: http://www.jove.com/video/50762/protocols-for-implementing-an-escherichia-coli-based-tx-tl-cell-free</h5> | <h5> For more information on TX-TL, see: http://www.jove.com/video/50762/protocols-for-implementing-an-escherichia-coli-based-tx-tl-cell-free</h5> | ||
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<h3>For any questions about Protocols, email: alverno.igem@gmail.com</h3> | <h3>For any questions about Protocols, email: alverno.igem@gmail.com</h3> |
Revision as of 19:55, 19 October 2016