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} | } | ||
+ | .accordion { | ||
+ | cursor: pointer; | ||
+ | padding: 18px; | ||
+ | width: 100%; | ||
+ | border: none; | ||
+ | text-align: left; | ||
+ | outline: none; | ||
+ | font-size: 17px; | ||
+ | transition: 0.4s; | ||
+ | color: #626262; | ||
+ | font: 400 0.875rem/1.375rem "Raleway", | ||
+ | } | ||
+ | |||
+ | .active, .accordion:hover { | ||
+ | background-color: #ccc; | ||
+ | } | ||
+ | |||
+ | .panel-body { | ||
+ | padding-top: 50px; | ||
+ | display: none; | ||
+ | background-color: white; | ||
+ | overflow: hidden; | ||
+ | } | ||
+ | |||
+ | .panel-body ol li{ | ||
+ | color: #626262; | ||
+ | font: 400 0.875rem/1.375rem "Raleway", | ||
+ | list-style-type: circle ; | ||
+ | } | ||
/**********************/ | /**********************/ | ||
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</div> | </div> | ||
</div> | </div> | ||
− | + | <div class="body-content" data-spy="scroll" data-target=".navbar" data-offset="50"> | |
− | + | <button class="accordion">Making Plates</button> | |
− | + | <div class="panel-body"> | |
− | + | <ol type="A"> | |
− | + | <li>Materials | |
− | + | <ol> | |
− | + | <li>Agar | |
− | + | <li>Antibiotic | |
− | + | <li>Empty plate | |
− | + | </li> | |
− | + | </ol> | |
− | + | <li>Procedure | |
− | + | <ol> | |
− | + | <li>Heat the agar for 15 minutes at power 4 in the microwave or until melted | |
− | + | <li>Retrieve empty plates and label with correct antibiotic | |
− | + | <li>Retrieve antibiotics and keep in ice, allow them to defrost | |
− | + | <li>Sterilize the area with ethanol/water mixture, and prepare a flame | |
− | + | <li>Add antibiotic to the container of agar | |
− | + | <ol type="a"> | |
− | + | <li>1000:1 ratio of agar:antibiotic | |
− | + | </li> | |
− | + | </ol> | |
− | + | <li>Flame containers and pour agar into plates | |
− | + | <ol type="a"> | |
− | + | <li>Just enough to cover bottom (around 5mL) | |
− | + | </li> | |
− | + | </ol> | |
− | + | <li>Remove any bubbles with flame | |
− | + | <li>Let cool and solidify, then place in fridge | |
− | + | </li> | |
− | + | </ol> | |
− | + | </li> | |
− | + | </ol> | |
− | + | </br> | |
− | + | </br> | |
− | + | </div> | |
− | + | <button class="accordion">Transformation (GeneHogs)</button> | |
− | + | <div class="panel-body"> | |
− | + | <ol type="A"> | |
− | + | <li>Materials | |
− | + | <ol> | |
− | + | <li>DNA Plasmid | |
− | + | <li>Plate with correct antibiotic | |
− | + | <li>Culture beads | |
− | + | <li>37 °C incubator | |
− | + | <li>37 °C shaker | |
− | + | <li>Hot water bath | |
− | + | <li>Ice bath | |
− | + | <li>LB media | |
− | + | <li>Microcentrifuge tubes | |
− | + | <li>GeneHogs cells | |
− | + | </li> | |
− | + | </ol> | |
− | + | <li>Procedure | |
− | + | <ol> | |
− | + | <li>Retrieve GeneHogs cells in ice bucket and allow them to thaw | |
− | + | <li>Resuspend DNA plasmid by adding 10μL of autoclaved nanowater to the correct well of the appropriate plate and then let sit for 5 minutes | |
− | + | <li>Prepare the heat-block water bath by filling a couple holes with water | |
− | + | <li>Sterilize the area and instruments with ethanol/water mixture, and prepare a flame | |
− | + | <li>Retrieve microcentrifuge tube and label them accordingly | |
− | + | <li>Transfer 50μL of GeneHogs and 1μL of DNA plasmid to the microcentrifuge tube | |
− | + | <li>Chill in ice for 20 minutes, then heat in the heat bath for 35 seconds, then chill again for 2 minutes | |
− | + | <li>Sterilize the area and instruments with ethanol/water mixture, and prepare a flame | |
− | + | <li>Add 200μL of LB broth into microcentrifuge tube | |
− | + | <li>Shake in the 37°C shaker for 1 hour | |
− | + | <li>Place the plate with the corresponding antibiotic in the 37°C incubator around the same time you place the microcentrifuge tube in the shaker | |
− | + | <li>Sterilize the area and instruments with ethanol/water mixture, and prepare a flame | |
− | + | <li>Pipette 100µL of the transformed cells onto the plate | |
− | + | <li>Spread around with beads, making sure to not have the beads circle the outside | |
− | + | <li>Dump beads in bleach and place plate in the 37°C incubator overnight | |
− | + | <li>Return the GenHog cells | |
− | + | </li> | |
− | + | </ol> | |
− | + | </li> | |
− | <li>Materials | + | </ol> |
− | <ol> | + | </br> |
− | + | </br> | |
− | <li>DNA Plasmid | + | </div> |
− | + | <button class="accordion">Inoculation</button> | |
− | <li>Plate with correct antibiotic | + | <div class="panel-body"> |
− | + | <ol type="A"> | |
− | <li>Culture beads | + | <li>Materials |
− | + | <ol> | |
− | <li>37 °C incubator | + | <li>Transformed colonie(s) |
− | + | <li>Corresponding antibiotic(s) | |
− | <li>37 °C shaker | + | <li>LB media |
− | + | <li>Wooden inoculation stick | |
− | <li>Hot water bath | + | <li>Plastic culture tube |
− | + | <li>37 °C shaker | |
− | <li>Ice bath | + | </li> |
− | + | </ol> | |
− | <li>LB media | + | <li>Procedure |
− | + | <ol> | |
− | <li>Microcentrifuge tubes | + | <li>Obtain LB broth, the correct antibiotic, and the plate with the desired colonies |
− | + | <li>Label plastic culture tube, create flame, and sterilize pipette, hands, tips, and bench | |
− | <li>GeneHogs cells | + | <li>Pipette 5mL LB into the culture tube |
− | </li> | + | <li>Add 5µL the antibiotic to the LB (1:1000 ratio of antibacterial to LB) |
− | </ol> | + | <li>Sterilize wooden stick |
− | + | <li>Grab a colony by touching the wooden stick to a lonely dot. | |
− | <li>Procedure | + | <li>Place the stick (colony-end) into the LB solution and shake well |
− | <ol> | + | <li>Shake at 250rpm in 37°C overnight |
− | + | <li>Return the LB broth, antibiotic, and plate | |
− | <li>Retrieve GeneHogs cells in ice bucket and allow them to thaw | + | </li> |
− | + | </ol> | |
− | <li>Resuspend DNA plasmid by adding 10μL of autoclaved nanowater to the correct well of the appropriate plate and then let sit for 5 minutes | + | </li> |
− | + | </ol> | |
− | <li>Prepare the heat-block water bath by filling a couple holes with water | + | </br> |
− | + | </br> | |
− | <li>Sterilize the area and instruments with ethanol/water mixture, and prepare a flame | + | </div> |
− | + | <button class="accordion">Miniprep (Zyppy Plasmid Miniprep Kit)</button> | |
− | <li>Retrieve microcentrifuge tube and label them accordingly | + | <div class="panel-body"> |
− | + | <ol type="A"> | |
− | <li>Transfer 50μL of GeneHogs and 1μL of DNA plasmid to the microcentrifuge tube | + | <li>Materials |
− | + | <ol> | |
− | <li>Chill in ice for 20 minutes, then heat in the heat bath for 35 seconds, then chill again for 2 minutes | + | <li>P1 buffer |
− | + | <li>P2 buffer | |
− | <li>Sterilize the area and instruments with ethanol/water mixture, and prepare a flame | + | <li>P3 buffer |
− | + | <li>Microcentrifuge Tubes | |
− | <li>Add 200μL of LB broth into microcentrifuge tube | + | <li>Filter Columns & Tubes |
− | + | <li>Endo-Wash Buffer | |
− | <li>Shake in the 37°C shaker for 1 hour | + | <li>Zippy-Wash Buffer |
− | + | <li>Autoclaved Nanowater | |
− | <li>Place the plate with the corresponding antibiotic in the 37°C incubator around the same time you place the microcentrifuge tube in the shaker | + | </li> |
− | + | </ol> | |
− | <li>Sterilize the area and instruments with ethanol/water mixture, and prepare a flame | + | <li>Procedure |
− | + | <ol> | |
− | <li>Pipette 100µL of the transformed cells onto the plate | + | <li>Ensure that the cultures are in a plastic culture tube. Place tubes in the centrifuge, balance the centrifuge, and run for 10 minutes at 5000 g |
− | + | <li>After centrifuging, dump out LB broth into broth waste and use a pipette to remove any remaining broth. Make sure not to touch the cells | |
− | <li>Spread around with beads, making sure to not have the beads circle the outside | + | <li>Pipette 200 µL of P1 buffer (Pink) to the culture tubes |
− | + | <li>After adding P1 buffer, pipette the solution up and down repeatedly to break up the pellet | |
− | <li>Dump beads in bleach and place plate in the 37°C incubator overnight | + | <ol type="a"> |
− | + | <li>Tilt the tube at an angle so the buffer isn’t covering the pellet. Use the pipette to extract the buffer and release it over the pellet | |
− | <li>Return the GenHog cells | + | </li> |
− | </li> | + | </ol> |
− | </ol> | + | <li>Transfer the buffer and pellet solution to appropriately labeled microcentrifuge tubes |
− | </li> | + | <li>Pipette 200 µL of P2 buffer (Blue) into the microcentrifuge tubes |
− | </ol> | + | <ol type="a"> |
− | + | <li>Invert microcentrifuge tubes twice | |
− | + | <li>Let sit for 1-2 minutes | |
− | + | </li> | |
− | + | </ol> | |
− | + | <li>Pipette 400 µL of P3 buffer (Yellow) into the microcentrifuge tubes | |
− | + | <ol type="a"> | |
− | + | <li>Obtain P3 buffer from the fridge and replace immediately after using | |
− | + | <li>Invert tubes repeatedly until the solution turns entirely yellow | |
− | + | </li> | |
− | + | </ol> | |
− | + | <li>Centrifuge tubes for 5 minutes at max speed | |
− | + | <ol type="a"> | |
− | + | <li>While centrifuging, set up filter column found in miniprep kit | |
− | + | </li> | |
− | + | </ol> | |
− | + | <li>Transfer supernatant from microcentrifuge tubes into filter tubes | |
− | + | <ol type="a"> | |
− | + | <li>Use a pipette to remove all of the supernatant, taking care not to touch any of the solid left in the microcentrifuge tubes | |
− | + | </li> | |
− | + | </ol> | |
− | + | <li>Centrifuge filter tubes for 30 seconds at 16,000 rpm | |
− | <li>Materials | + | <ol type="a"> |
− | <ol> | + | <li>Discard the flowthrough in the biohazard waste |
− | + | </li> | |
− | <li>Transformed colonie(s) | + | </ol> |
− | + | <li>Add 200 µL of the Endo-Wash buffer to the filter tubes and centrifuge at 16,000 rpm for 30 seconds | |
− | <li>Corresponding antibiotic(s) | + | <ol type="a"> |
− | + | <li>Discard the flowthrough | |
− | <li>LB media | + | </li> |
− | + | </ol> | |
− | <li>Wooden inoculation stick | + | <li>Add 400 µL of the Zippy-Wash buffer to the filter tubes and centrifuge at 16,000 rpm for 30 seconds |
− | + | <li>Centrifuge for an additional 2 minutes at 20,000 g | |
− | <li>Plastic culture tube | + | <ol type="a"> |
− | + | <li>Discard flowthrough | |
− | <li>37 °C shaker | + | </li> |
− | </li> | + | </ol> |
− | </ol> | + | <li>Label new microcentrifuge tubes with the plasmid name |
− | + | <ol type="a"> | |
− | <li>Procedure | + | <li>Plasmid: pSB3K3 & pSB4A5 |
− | <ol> | + | </li> |
− | + | </ol> | |
− | <li>Obtain LB broth, the correct antibiotic, and the plate with the desired colonies | + | <li>Place filter tubes into new microcentrifuge tubes |
− | + | <li>Add 30 µL of autoclaved Nanowater to the filter tubes | |
− | <li>Label plastic culture tube, create flame, and sterilize pipette, hands, tips, and bench | + | <ol type="a"> |
− | + | <li>Let stand for 1 minute | |
− | <li>Pipette 5mL LB into the culture tube | + | </li> |
− | + | </ol> | |
− | <li>Add 5µL the antibiotic to the LB (1:1000 ratio of antibacterial to LB) | + | <li>Centrifuge filter tubes for 1.5 minutes at 16,000 g |
− | + | <li>Find DNA concentration(X) using NanoDrop | |
− | <li>Sterilize wooden stick | + | </li> |
− | + | </ol> | |
− | <li>Grab a colony by touching the wooden stick to a lonely dot. | + | </ol> |
− | + | </br> | |
− | <li>Place the stick (colony-end) into the LB solution and shake well | + | </br> |
− | + | </div> | |
− | <li>Shake at 250rpm in 37°C overnight | + | <button class="accordion">Digestion</button> |
− | + | <div class="panel-body"> | |
− | <li>Return the LB broth, antibiotic, and plate | + | <ol type="A"> |
− | + | <li>Materials | |
− | </li> | + | <ol> |
− | </ol> | + | <li>PCR Tubes |
− | </li> | + | <li>EcoR1 |
− | </ol> | + | <li>Pst1 |
− | + | <li>Cutsmart Buffer | |
− | + | </li> | |
− | + | </ol> | |
− | + | <li>Procedure | |
− | + | <ol> | |
− | + | <li>Obtain and label PCR tubes | |
− | + | <li>Add the following components to the tube: | |
− | + | <ol type="a"> | |
− | + | <li>1 µL plasmid | |
− | + | <ol type="I"> | |
− | + | <li>Calculate the volume of plasmid required for 1 µg | |
− | + | </ol> | |
− | + | <li>1 µL of EcoR1 | |
− | + | <li>1 µL of Pst1 | |
− | + | <li>5 µL of Cutsmart buffer | |
− | + | <li>X µL of autoclaved Nanowater | |
− | + | <ol type="I"> | |
− | + | <li>X = water required to bring the total volume to 50 µL | |
− | + | </li> | |
− | <li>Materials | + | </ol> |
− | <ol> | + | </li> |
− | + | </ol> | |
− | <li>P1 buffer | + | <li>Set tube in thermal cycler |
− | + | <ol type="a"> | |
− | <li>P2 buffer | + | <li>Run using “Digest” setting---37 degrees Celsius, 3 hours |
− | + | </li> | |
− | <li>P3 buffer | + | </ol> |
− | + | </li> | |
− | <li>Microcentrifuge Tubes | + | </ol> |
− | + | <li>Calculations | |
− | <li>Filter Columns & Tubes | + | <ol> |
− | + | <li> | |
− | <li>Endo-Wash Buffer | + | 1000 ng * <sup>1 L</sup>⁄<sub>x ng</sub> = µL plasmid |
− | + | <br/>X = concentration of plasmid | |
− | <li>Zippy-Wash Buffer | + | </li> |
− | + | </ol> | |
− | <li>Autoclaved Nanowater | + | </li> |
− | </li> | + | </ol> |
− | </ol> | + | </br> |
− | + | </br> | |
− | <li>Procedure | + | </div> |
− | <ol> | + | <button class="accordion">Gel Imaging</button> |
− | + | <div class="panel-body"> | |
− | <li>Ensure that the cultures are in a plastic culture tube. Place tubes in the centrifuge, balance the centrifuge, and run for 10 minutes at 5000 g | + | <ol type="A"> |
− | + | <li>Materials | |
− | + | <ol> | |
− | <li>After centrifuging, dump out LB broth into broth waste and use a pipette to remove any remaining broth. Make sure not to touch the cells | + | <li>1x TAE Buffer |
− | + | <li>Agarose | |
− | <li>Pipette 200 µL of P1 buffer (Pink) to the culture tubes | + | <li>Ethidium Bromide |
− | + | <li>Gel Holder/ Well comb | |
− | <li>After adding P1 buffer, pipette the solution up and down repeatedly to break up the pellet | + | <li>Electrophoresis Machine |
− | <ol> | + | <li>DNA ladder |
− | + | <li>DNA dye | |
− | <li>Tilt the tube at an angle so the buffer isn’t covering the pellet. Use the pipette to extract the buffer and release it over the pellet | + | <li>Imaging Apparatus/Software |
− | </li> | + | </li> |
− | </ol> | + | </ol> |
− | + | <li>Procedure | |
− | <li>Transfer the buffer and pellet solution to appropriately labeled microcentrifuge tubes | + | <ol> |
− | + | <li>Add 50 mL of 1x TAE and 0.75 g of agarose to the correct flask | |
− | <li>Pipette 200 µL of P2 buffer (Blue) into the microcentrifuge tubes | + | <ol type="a"> |
− | <ol> | + | <li>Place a smaller flask upside down inside the larger flask, covering it |
− | + | </li> | |
− | <li>Invert microcentrifuge tubes twice | + | </ol> |
− | + | <li>Place flask in microwave for 1.5 minutes at power level 10 | |
− | <li>Let sit for 1-2 minutes | + | <li>Allow agarose to cool for 10 minutes |
− | </li> | + | <li>Add 4 µL of ethidium bromide inside the fume hood |
− | </ol> | + | <ol type="a"> |
− | + | <li>Ethidium bromide is a carcinogen; do not touch or inhale | |
− | <li>Pipette 400 µL of P3 buffer (Yellow) into the microcentrifuge tubes | + | <li>Dispose of all tips in the ethidium bromide tip container |
− | <ol> | + | </li> |
− | + | </ol> | |
− | <li>Obtain P3 buffer from the fridge and replace immediately after using | + | <li>Pour agarose into a gel holder and place the appropriately-sized well comb |
− | + | <ol type="a"> | |
− | <li>Invert tubes repeatedly until the solution turns entirely yellow | + | <li>Pop any bubbles in the agarose |
− | </li> | + | <li>Allow to cool for 20 minutes |
− | </ol> | + | <li>Insert the gel tray into the gel box with the wells close to the black electrode |
− | + | </li> | |
− | <li>Centrifuge tubes for 5 minutes at max speed | + | </ol> |
− | <ol> | + | <li>Use arrow alongside gel box to orient tray if needed; DNA will move from the black electrode to the red electrode |
− | + | <ol type="a"> | |
− | <li>While centrifuging, set up filter column found in miniprep kit | + | <li>“Run to red” |
− | </li> | + | <li>Pour enough 1x TAE to cover the gel and reach the max fill line |
− | </ol> | + | <li>Choose your DNA ladder based on how large you expect the DNA fragments to be |
− | + | <li>Obtain DNA ladder from fridge | |
− | <li>Transfer supernatant from microcentrifuge tubes into filter tubes | + | <li>Obtain gel loading dye from tray near electrophoresis equipment |
− | <ol> | + | </li> |
− | + | </ol> | |
− | <li>Use a pipette to remove all of the supernatant, taking care not to touch any of the solid left in the microcentrifuge tubes | + | <li>Note: both the DNA ladder and the loading dye are purple. Do not confuse them! |
− | </li> | + | <ol type="a"> |
− | </ol> | + | <li>Add loading dye to the DNA sample |
− | + | </li> | |
− | <li>Centrifuge filter tubes for 30 seconds at 16,000 rpm | + | </ol> |
− | <ol> | + | <li>Add dye to DNA in a 1:6 dilution |
− | + | <ol type="a"> | |
− | <li>Discard the flowthrough in the biohazard waste | + | <li>For example, add 10 µL of dye to 50 µL of DNA |
− | </li> | + | <li>Pipette the DNA/dye solution up and down to mix |
− | </ol> | + | <li>Add 8 µL of the ladder to well 1 |
− | + | <li>Place up to 30 µL of DNA into wells 2-8 as needed | |
− | <li>Add 200 µL of the Endo-Wash buffer to the filter tubes and centrifuge at 16,000 rpm for 30 seconds | + | <li>Place lid on gel box and connect to a volt machine. |
− | <ol> | + | </li> |
− | + | </ol> | |
− | <li>Discard the flowthrough | + | <li>Set to 100 volts for 60 minutes and hit run |
− | </li> | + | <ol type="a"> |
− | </ol> | + | <li>Bubbles should appear if the electrophoresis is working |
− | + | </li> | |
− | <li>Add 400 µL of the Zippy-Wash buffer to the filter tubes and centrifuge at 16,000 rpm for 30 seconds | + | </ol> |
− | + | <li>Time depends on the size of DNA fragments. Check gel periodically to make sure the DNA does not run off the end of the gel | |
− | <li>Centrifuge for an additional 2 minutes at 20,000 g | + | <ol type="a"> |
− | <ol> | + | <li>Place gel in ChemiDoc |
− | + | <li>Open ImageLab3 Software | |
− | <li>Discard flowthrough | + | <li>Select ethidium bromide |
− | </li> | + | <li>Adjust filter based on instructions from the software |
− | </ol> | + | <li>Use Image Tools to crop the picture |
− | + | <li>Adjust contrast to better see the gel if desired | |
− | <li>Label new microcentrifuge tubes with the plasmid name | + | <li>Use Annotation Tools to create notes |
− | <ol> | + | <li>Ladder type |
− | + | <li>Digestion product | |
− | <li>Plasmid: pSB3K3 & pSB4A5 | + | <li>Digestion enzyme names |
− | </li> | + | <li>Analyze gel to determine if the gene were successfully excised from the plasmid |
− | </ol> | + | </li> |
− | + | </ol> | |
− | <li>Place filter tubes into new microcentrifuge tubes | + | </li> |
− | + | </ol> | |
− | <li>Add 30 µL of autoclaved Nanowater to the filter tubes | + | </li> |
− | <ol> | + | </ol> |
− | + | </br> | |
− | <li>Let stand for 1 minute | + | </br> |
− | </li> | + | </div> |
− | </ol> | + | <button class="accordion">Gel Recovery</button> |
− | + | <div class="panel-body"> | |
− | <li>Centrifuge filter tubes for 1.5 minutes at 16,000 g | + | <ol type="A"> |
− | + | <li>Materials | |
− | <li>Find DNA concentration(X) using NanoDrop | + | <ol> |
− | </li> | + | <li>Gel Recovery Kit |
− | </ol> | + | <li>Centrifuge |
− | + | <li>Microcentrifuge Tubes | |
− | </ol> | + | <li>Autoclaved Nanowater |
− | + | <li>Nanodrop | |
− | + | </li> | |
− | + | </ol> | |
− | + | <li>Procedure | |
− | + | <ol> | |
− | + | <li>Use UV lamp with face shields to visualize bands in gel | |
− | + | <li>Use razor to carefully cut the bands out of the gel | |
− | + | <ol type="a"> | |
− | </ | + | <li>Store them in their own microcentrifuge tubes (labeled) |
− | + | </li> | |
− | + | </ol> | |
− | + | <li>Dispose of excess gel and gloves in special container | |
− | + | <ol type="a"> | |
− | + | <li>This is due to the ethidium bromide | |
− | + | </li> | |
− | + | </ol> | |
− | + | <li>Determine and record the weight of the recovered gels | |
− | + | <ol type="a"> | |
− | + | <li>Use an empty microcentrifuge tube to tare the balance | |
− | + | </li> | |
− | + | </ol> | |
− | + | <li>Retrieve the DNA Recovery Kit | |
− | + | <li>Add X amount of Agarose dissolving buffer(ADB) to the respective microcentrifuge tube | |
− | + | <ol type="a"> | |
− | + | <li>X = 3000 * (gel weight) | |
− | + | <ol type="I"> | |
− | + | <li>EX: 3000 * (0.126 g) = 378 µL | |
− | + | </li> | |
− | + | </ol> | |
− | + | <li>The max amount of ADB that can be added is 750 µL. If the gel weighs more than 0.250 g, then more gel must be cut off or the gel must be divided into two separate tubes | |
− | + | </li> | |
− | <li>Materials | + | </ol> |
− | <ol> | + | <li>Place the microcentrifuge tubes in a 50-55 °C water bath for 10 minutes |
− | + | <ol type="a"> | |
− | <li>PCR Tubes | + | <li>Make sure the gel has completely dissolved, if there are pieces present, put back in the water bath for a couple minutes |
− | + | </li> | |
− | <li>EcoR1 | + | </ol> |
− | + | <li>Vortex to mix well | |
− | <li>Pst1 | + | <li>Retrieve and label collection tubes and their filter counterparts |
− | + | <li>Transfer the liquid into the filter using a pipette | |
− | <li>Cutsmart Buffer | + | <li>Centrifuge for 30 s at 16,000 G |
− | </li> | + | <ol type="a"> |
− | </ol> | + | <li>Discard flowthrough |
− | + | </li> | |
− | <li>Procedure | + | </ol> |
− | <ol> | + | <li>Add 200 µL of wash buffer into the filter and centrifuge again with the same conditions |
− | + | <li>Repeat the wash buffer step | |
− | <li>Obtain and label PCR tubes | + | <li>Centrifuge again for a minute at 21,000 G |
− | + | <li>Move the filters into new, labeled microcentrifuge tubes | |
− | <li>Add the following components to the tube: | + | <li>Add 10 µL of autoclaved nanowater and centrifuge for 1 min at 16,000 G |
− | <ol> | + | <li>Check and record the concentration with the nanodrop machine |
− | + | </li> | |
− | <li>1 µL plasmid | + | </ol> |
− | <ol> | + | </li> |
− | + | </ol> | |
− | <li>Calculate the volume of plasmid required for 1 µg | + | </br> |
− | + | </br> | |
− | + | </div> | |
− | + | <button class="accordion">Fluorescence Characterizations</button> | |
− | + | <div class="panel-body"> | |
− | + | <ol type="A"> | |
− | + | <li>Materials | |
− | </ol> | + | <ol> |
− | + | <li>BBa_K584000 cells | |
− | + | <li>BBa_K206000 cells | |
− | + | <li>L-arabinose | |
− | <li>1 µL of EcoR1 | + | <li>Autoclaved NanoWater |
− | + | <li>LB Media | |
− | <li>1 µL of Pst1 | + | <li>1x PBS |
− | + | <li>Centrifuge | |
− | <li>5 µL of Cutsmart buffer | + | <li>Plastic culture tubes |
− | + | <li>Plate reader | |
− | <li>X µL of autoclaved Nanowater | + | <li>Flow cytometer |
− | <ol> | + | </li> |
− | + | ||
− | <li>X = water required to bring the total volume to 50 µL | + | |
− | </li> | + | |
− | </ol> | + | |
− | </li> | + | |
− | </ol> | + | |
− | + | ||
− | <li>Set tube in thermal cycler | + | |
− | <ol> | + | |
− | + | ||
− | <li>Run using “Digest” setting---37 degrees Celsius, 3 hours | + | |
− | </li> | + | |
− | </ol> | + | |
− | </li> | + | |
− | </ol> | + | |
− | < | + | |
− | < | + | |
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− | < | + | |
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− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | <ol> | + | |
− | + | ||
− | <li>Materials | + | |
− | <ol> | + | |
− | + | ||
− | <li>1x TAE Buffer | + | |
− | + | ||
− | <li>Agarose | + | |
− | + | ||
− | <li>Ethidium Bromide | + | |
− | + | ||
− | <li>Gel Holder/ Well comb | + | |
− | + | ||
− | <li>Electrophoresis Machine | + | |
− | + | ||
− | <li>DNA ladder | + | |
− | + | ||
− | <li>DNA dye | + | |
− | + | ||
− | <li>Imaging Apparatus/Software | + | |
− | </li> | + | |
− | </ol> | + | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | <li>Procedure | + | |
− | <ol> | + | |
− | + | ||
− | <li>Add 50 mL of 1x TAE and 0.75 g of agarose to the correct flask | + | |
− | <ol> | + | |
− | + | ||
− | <li>Place a smaller flask upside down inside the larger flask, covering it | + | |
− | </li> | + | |
− | </ol> | + | |
− | + | ||
− | <li>Place flask in microwave for 1.5 minutes at power level 10 | + | |
− | + | ||
− | <li>Allow agarose to cool for 10 minutes | + | |
− | + | ||
− | <li>Add 4 µL of ethidium bromide inside the fume hood | + | |
− | <ol> | + | |
− | + | ||
− | <li>Ethidium bromide is a carcinogen; do not touch or inhale | + | |
− | + | ||
− | <li>Dispose of all tips in the ethidium bromide tip container | + | |
− | </li> | + | |
− | </ol> | + | |
− | + | ||
− | <li>Pour agarose into a gel holder and place the appropriately-sized well comb | + | |
− | <ol> | + | |
− | + | ||
− | <li>Pop any bubbles in the agarose | + | |
− | + | ||
− | <li>Allow to cool for 20 minutes | + | |
− | + | ||
− | <li>Insert the gel tray into the gel box with the wells close to the black electrode | + | |
− | </li> | + | |
− | </ol> | + | |
− | + | ||
− | <li>Use arrow alongside gel box to orient tray if needed; DNA will move from the black electrode to the red electrode | + | |
− | <ol> | + | |
− | + | ||
− | <li>“Run to red” | + | |
− | + | ||
− | <li>Pour enough 1x TAE to cover the gel and reach the max fill line | + | |
− | + | ||
− | <li>Choose your DNA ladder based on how large you expect the DNA fragments to be | + | |
− | + | ||
− | <li>Obtain DNA ladder from fridge | + | |
− | + | ||
− | <li>Obtain gel loading dye from tray near electrophoresis equipment | + | |
− | </li> | + | |
− | </ol> | + | |
− | + | ||
− | <li>Note: both the DNA ladder and the loading dye are purple. Do not confuse them! | + | |
− | <ol> | + | |
− | + | ||
− | <li>Add loading dye to the DNA sample | + | |
− | </li> | + | |
− | </ol> | + | |
− | + | ||
− | <li>Add dye to DNA in a 1:6 dilution | + | |
− | <ol> | + | |
− | + | ||
− | <li>For example, add 10 µL of dye to 50 µL of DNA | + | |
− | + | ||
− | <li>Pipette the DNA/dye solution up and down to mix | + | |
− | + | ||
− | <li>Add 8 µL of the ladder to well 1 | + | |
− | + | ||
− | <li>Place up to 30 µL of DNA into wells 2-8 as needed | + | |
− | + | ||
− | <li>Place lid on gel box and connect to a volt machine. | + | |
− | </li> | + | |
− | </ol> | + | |
− | + | ||
− | <li>Set to 100 volts for 60 minutes and hit run | + | |
− | + | ||
− | <li>Bubbles should appear if the electrophoresis is working | + | |
− | + | ||
− | <li>Time depends on the size of DNA fragments. Check gel periodically to make sure the DNA does not run off the end of the gel | + | |
− | <ol> | + | |
− | + | ||
− | <li>Place gel in ChemiDoc | + | |
− | + | ||
− | <li>Open ImageLab3 Software | + | |
− | + | ||
− | <li>Select ethidium bromide | + | |
− | + | ||
− | <li>Adjust filter based on instructions from the software | + | |
− | + | ||
− | <li>Use Image Tools to crop the picture | + | |
− | + | ||
− | <li>Adjust contrast to better see the gel if desired | + | |
− | + | ||
− | <li>Use Annotation Tools to create notes | + | |
− | + | ||
− | + | ||
− | + | ||
− | <li>Ladder type | + | |
− | + | ||
− | <li>Digestion product | + | |
− | + | ||
− | <li>Digestion enzyme names | + | |
− | + | ||
− | <li>Analyze gel to determine if the gene were successfully excised from the plasmid | + | |
− | </li> | + | |
− | </ol> | + | |
− | </li> | + | |
− | </ol> | + | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | <div | + | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | <li>Materials | + | |
− | <ol> | + | |
− | + | ||
− | <li>Gel Recovery Kit | + | |
− | + | ||
− | <li>Centrifuge | + | |
− | + | ||
− | <li>Microcentrifuge Tubes | + | |
− | + | ||
− | <li>Autoclaved Nanowater | + | |
− | + | ||
− | <li>Nanodrop | + | |
− | </li> | + | |
− | </ol> | + | |
− | + | ||
− | <li>Procedure | + | |
− | <ol> | + | |
− | + | ||
− | <li>Use UV lamp with face shields to visualize bands in gel | + | |
− | + | ||
− | <li>Use razor to carefully cut the bands out of the gel | + | |
− | <ol> | + | |
− | + | ||
− | <li>Store them in their own microcentrifuge tubes (labeled) | + | |
− | </li> | + | |
− | </ol> | + | |
− | + | ||
− | <li>Dispose of excess gel and gloves in special container | + | |
− | <ol> | + | |
− | + | ||
− | <li>This is due to the ethidium bromide | + | |
− | </li> | + | |
− | </ol> | + | |
− | + | ||
− | <li>Determine and record the weight of the recovered gels | + | |
− | <ol> | + | |
− | + | ||
− | <li>Use an empty microcentrifuge tube to tare the balance | + | |
− | </li> | + | |
− | </ol> | + | |
− | + | ||
− | <li>Retrieve the DNA Recovery Kit | + | |
− | + | ||
− | <li>Add X amount of Agarose dissolving buffer(ADB) to the respective microcentrifuge tube | + | |
− | <ol> | + | |
− | + | ||
− | <li>X = 3000 * (gel weight) | + | |
− | <ol> | + | |
− | + | ||
− | <li>EX: 3000 * (0.126 g) = 378 µL | + | |
− | </li> | + | |
− | </ol> | + | |
− | + | ||
− | <li>The max amount of ADB that can be added is 750 µL. If the gel weighs more than 0.250 g, then more gel must be cut off or the gel must be divided into two separate tubes | + | |
− | </li> | + | |
− | </ol> | + | |
− | + | ||
− | <li>Place the microcentrifuge tubes in a 50-55 °C water bath for 10 minutes | + | |
− | <ol> | + | |
− | + | ||
− | <li>Make sure the gel has completely dissolved, if there are pieces present, put back in the water bath for a couple minutes | + | |
− | </li> | + | |
− | </ol> | + | |
− | + | ||
− | <li>Vortex to mix well | + | |
− | + | ||
− | <li>Retrieve and label collection tubes and their filter counterparts | + | |
− | + | ||
− | <li>Transfer the liquid into the filter using a pipette | + | |
− | + | ||
− | <li>Centrifuge for 30 s at 16,000 G | + | |
− | <ol> | + | |
− | + | ||
− | <li>Discard flowthrough | + | |
− | </li> | + | |
− | </ol> | + | |
− | + | ||
− | <li>Add 200 µL of wash buffer into the filter and centrifuge again with the same conditions | + | |
− | + | ||
− | <li>Repeat the wash buffer step | + | |
− | + | ||
− | <li>Centrifuge again for a minute at 21,000 G | + | |
− | + | ||
− | <li>Move the filters into new, labeled microcentrifuge tubes | + | |
− | + | ||
− | <li>Add 10 µL of autoclaved nanowater and centrifuge for 1 min at 16,000 G | + | |
− | + | ||
− | <li>Check and record the concentration with the nanodrop machine | + | |
− | </li> | + | |
− | </ol> | + | |
− | </li> | + | |
− | </ol> | + | |
− | + | ||
− | + | ||
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− | + | ||
− | + | ||
− | + | ||
− | <li>Materials | + | |
− | <ol> | + | |
− | + | ||
− | <li>BBa_K584000 cells | + | |
− | + | ||
− | <li>BBa_K206000 cells | + | |
− | + | ||
− | <li>L-arabinose | + | |
− | + | ||
− | <li>Autoclaved NanoWater | + | |
− | + | ||
− | <li>LB Media | + | |
− | + | ||
− | <li>1x PBS | + | |
− | + | ||
− | <li>Centrifuge | + | |
− | + | ||
− | <li>Plastic culture tubes | + | |
− | + | ||
− | <li>Plate reader | + | |
− | + | ||
− | <li>Flow cytometer | + | |
− | </li> | + | |
</ol> | </ol> | ||
Line 984: | Line 698: | ||
<li>Perform a dilution for each sample into 10 individual tubes (triplicates of 3 conditions per sample and 1 tube to monitor OD growth) with more LB to OD<sub>600</sub> 0.05 and let culture until 0.1. The new volume should be 1 mL. There should be 20 tubes total. Antibiotics should be added also. | <li>Perform a dilution for each sample into 10 individual tubes (triplicates of 3 conditions per sample and 1 tube to monitor OD growth) with more LB to OD<sub>600</sub> 0.05 and let culture until 0.1. The new volume should be 1 mL. There should be 20 tubes total. Antibiotics should be added also. | ||
− | <ol> | + | <ol type="a"> |
<li>(OD1)(x)=(0.05)(1mL) | <li>(OD1)(x)=(0.05)(1mL) | ||
Line 991: | Line 705: | ||
x = volume from culture | x = volume from culture | ||
</p> | </p> | ||
− | <ol> | + | </li> |
+ | </ol> | ||
<li>After OD<sub>600</sub> is 0.1, use 500 µL from each of the two cultures to find the starting OD<sub>595</sub> and fluorescence with the plate reader | <li>After OD<sub>600</sub> is 0.1, use 500 µL from each of the two cultures to find the starting OD<sub>595</sub> and fluorescence with the plate reader | ||
− | <ol> | + | <ol type="a"> |
<li>Use a clear plate to measure OD<sub>595 </sub>and a black plate to measure fluorescence | <li>Use a clear plate to measure OD<sub>595 </sub>and a black plate to measure fluorescence | ||
<li>Excitation & emission - <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_E0040">http://parts.igem.org/wiki/index.php?title=Part:BBa_E004</a> | <li>Excitation & emission - <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_E0040">http://parts.igem.org/wiki/index.php?title=Part:BBa_E004</a> | ||
− | <ol> | + | <ol type="I"> |
<li>490 nm excitation | <li>490 nm excitation | ||
Line 1,010: | Line 725: | ||
<li>Wash and resuspend cells in PBS for initial measurement | <li>Wash and resuspend cells in PBS for initial measurement | ||
− | <ol> | + | <ol type="a"> |
− | <li> | + | <li>Cool centrifuge to 4 °C, use plastic culture tubes, and centrifuge at 5,000 g for 10 minutes |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
<li>Discard supernatant | <li>Discard supernatant | ||
Line 1,027: | Line 736: | ||
</ol> | </ol> | ||
− | <li>Add 5 mL of the correct culture into each of the 6 culture tubes (BBa_K584000 should be in 3 of the tubes, BBa_K206000 in 3) Add culture, 20% L-arabinose, and/or autoclaved Nanowater in the following proportions: | + | <li>Add 5 mL of the correct culture into each of the 6 culture tubes (BBa_K584000 should be in 3 of the tubes, BBa_K206000 in the other 3) Add culture, 20% L-arabinose, and/or autoclaved Nanowater in the following proportions: |
− | <ol> | + | <ol type="a"> |
<li>0% | <li>0% | ||
− | <ol> | + | <ol type="I"> |
<li>5 mL BBa_K584000/BBa_K206000 | <li>5 mL BBa_K584000/BBa_K206000 | ||
Line 1,040: | Line 749: | ||
<li>0.2% | <li>0.2% | ||
− | <ol> | + | <ol type="I"> |
<li>5 ml BBa_K584000/BBa_K206000 | <li>5 ml BBa_K584000/BBa_K206000 | ||
Line 1,051: | Line 760: | ||
<li>2% | <li>2% | ||
− | <ol> | + | <ol type="I"> |
<li>5 mL BBa_K584000/BBa_K206000 | <li>5 mL BBa_K584000/BBa_K206000 | ||
Line 1,064: | Line 773: | ||
<li>Remove 500 µL from each culture after 1 hour and wash and resuspend with PBS | <li>Remove 500 µL from each culture after 1 hour and wash and resuspend with PBS | ||
− | <ol> | + | <ol type="a"> |
− | + | <li>Cool centrifuge to 4 °C, use plastic culture tubes, and centrifuge at 5,000 g for 10 minutes | |
− | + | ||
− | + | ||
− | <li>Cool centrifuge to 4 °C | + | |
− | + | ||
− | + | ||
</li> | </li> | ||
</ol> | </ol> | ||
Line 1,078: | Line 782: | ||
<li>Add 500 µL of PBS and resuspend | <li>Add 500 µL of PBS and resuspend | ||
− | + | ||
− | + | ||
− | + | ||
<li>Use 100 µL from each of the cultures to find the OD<sub>595</sub> and fluorescence with the plate reader | <li>Use 100 µL from each of the cultures to find the OD<sub>595</sub> and fluorescence with the plate reader | ||
<li>Save 300 µL from each culture for flow cytometry | <li>Save 300 µL from each culture for flow cytometry | ||
− | <ol> | + | <ol type="a"> |
<li>Place 300 µL sample in 4 °C refrigerator to halt growth | <li>Place 300 µL sample in 4 °C refrigerator to halt growth | ||
Line 1,100: | Line 802: | ||
<li>BBa_K206000 Culture Dilution | <li>BBa_K206000 Culture Dilution | ||
− | <ol> | + | <ol type="a"> |
<li> | <li> | ||
Line 1,106: | Line 808: | ||
</li> | </li> | ||
</ol> | </ol> | ||
− | + | ||
− | + | ||
<li>BBa_K584000 Culture Dilution | <li>BBa_K584000 Culture Dilution | ||
− | <ol> | + | <ol type="a"> |
<li> | <li> | ||
Line 1,128: | Line 829: | ||
</li> | </li> | ||
</ol> | </ol> | ||
− | + | </br> | |
− | + | </br> | |
− | + | </div> | |
− | + | <button class="accordion">Glycerol Stock</button> | |
− | + | <div class="panel-body"> | |
− | + | <ol type="A"> | |
− | + | <li>Materials | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | <li>Materials | + | |
<ol> | <ol> | ||
Line 1,159: | Line 848: | ||
<li>Obtain glycerol tubes and label appropriately | <li>Obtain glycerol tubes and label appropriately | ||
− | <ol> | + | <ol type="a"> |
<li>pBAD + CheZ #1 7/24/19 | <li>pBAD + CheZ #1 7/24/19 | ||
Line 1,180: | Line 869: | ||
</li> | </li> | ||
</ol> | </ol> | ||
− | + | </br> | |
− | + | </br> | |
− | + | </div> | |
− | + | <button class="accordion">LB Broth Preparation</button> | |
− | + | <div class="panel-body"> | |
− | + | <ol type="A"> | |
− | + | <li>Materials | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | <li>Materials | + | |
<ol> | <ol> | ||
Line 1,219: | Line 897: | ||
</ol> | </ol> | ||
</li> | </li> | ||
− | </ol> | + | </ol> |
− | + | </br> | |
− | + | </br> | |
− | + | </div> | |
− | + | <button class="accordion">TB Broth Preparation</button> | |
− | + | <div class="panel-body"> | |
− | + | <ol type="A"> | |
− | + | <li>Materials | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
<ol> | <ol> | ||
Line 1,261: | Line 927: | ||
<li>Add the following quantities together in a 250 mL bottle for TB: | <li>Add the following quantities together in a 250 mL bottle for TB: | ||
− | <ol> | + | <ol type="a"> |
<li>Yeast Extract: 6 g | <li>Yeast Extract: 6 g | ||
Line 1,276: | Line 942: | ||
<li>Add the following quantities together in a 250 mL bottle for LB broth: | <li>Add the following quantities together in a 250 mL bottle for LB broth: | ||
− | <ol> | + | <ol type="a"> |
<li>LB broth powder: 6.25 g | <li>LB broth powder: 6.25 g | ||
Line 1,285: | Line 951: | ||
<li>Add the following quantities together in a 500 mL bottle for LB agar: | <li>Add the following quantities together in a 500 mL bottle for LB agar: | ||
− | <ol> | + | <ol type="a"> |
<li>LB broth powder: 12.5 g | <li>LB broth powder: 12.5 g | ||
Line 1,302: | Line 968: | ||
</li> | </li> | ||
</ol> | </ol> | ||
− | + | </br> | |
− | + | </br> | |
− | + | </div> | |
− | + | <button class="accordion">Polymerase Chain Reaction (PCR)</button> | |
− | + | <div class="panel-body"> | |
− | + | <ol type="A"> | |
− | + | <li>Materials | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
<ol> | <ol> | ||
Line 1,344: | Line 999: | ||
<li>Create the primer mixtures by combining the following components: | <li>Create the primer mixtures by combining the following components: | ||
− | <ol> | + | <ol type="a"> |
<li>1 μL forward primer | <li>1 μL forward primer | ||
Line 1,355: | Line 1,010: | ||
<li>Create DNA templates by combining the following components: | <li>Create DNA templates by combining the following components: | ||
− | <ol> | + | <ol type="a"> |
<li>1 μL stock pBAD+GarKS+NSP4 template (100 ng/μL) | <li>1 μL stock pBAD+GarKS+NSP4 template (100 ng/μL) | ||
Line 1,364: | Line 1,019: | ||
<li>Create a 50 μL PCR mixture by adding the following components to a PCR tube: | <li>Create a 50 μL PCR mixture by adding the following components to a PCR tube: | ||
− | <ol> | + | <ol type="a"> |
<li>32.5 μL autoclaved Nanowater | <li>32.5 μL autoclaved Nanowater | ||
Line 1,379: | Line 1,034: | ||
<li>1 μL KOD Polymerase | <li>1 μL KOD Polymerase | ||
− | <ol> | + | <ol type="I"> |
<li>Be sure to add the KOD polymerase last; the order of the other components can be changed | <li>Be sure to add the KOD polymerase last; the order of the other components can be changed | ||
− | + | ||
− | + | ||
</li> | </li> | ||
</ol> | </ol> | ||
Line 1,392: | Line 1,046: | ||
<li>Modify the time for the 70 °C cycle to match the length of the template | <li>Modify the time for the 70 °C cycle to match the length of the template | ||
− | <ol> | + | <ol type="a"> |
<li>Set time to 18 seconds for the two garvicin parts | <li>Set time to 18 seconds for the two garvicin parts | ||
Line 1,403: | Line 1,057: | ||
</li> | </li> | ||
</ol> | </ol> | ||
− | + | </br> | |
− | + | </br> | |
− | + | </div> | |
− | + | <button class="accordion">Gibson Cloning</button> | |
− | + | <div class="panel-body"> | |
− | + | <ol type="A"> | |
− | + | <li>Materials | |
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<ol> | <ol> | ||
Line 1,454: | Line 1,096: | ||
<li>Obtain parts, vector, nanowater, master mix, and PCR tubes | <li>Obtain parts, vector, nanowater, master mix, and PCR tubes | ||
− | <ol> | + | <ol type="a"> |
<li>Thaw and vortex master mix thoroughly and keep on ice | <li>Thaw and vortex master mix thoroughly and keep on ice | ||
Line 1,464: | Line 1,106: | ||
<li>Dilute each part with 10 µL of autoclaved nanowater to make an approximate concentration of 100 ng/µL | <li>Dilute each part with 10 µL of autoclaved nanowater to make an approximate concentration of 100 ng/µL | ||
− | <li>Add the following quantities to the PCR | + | <li>Add the following quantities to the PCR tube (total of 20 µL): |
− | <ol> | + | <ol type="a"> |
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<li>Insert: x | <li>Insert: x | ||
Line 1,478: | Line 1,118: | ||
<li>HiFi DNA Assembly Master Mix: 10 µL | <li>HiFi DNA Assembly Master Mix: 10 µL | ||
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− | |||
</li> | </li> | ||
</ol> | </ol> | ||
+ | <li> Incubate samples in a thermocycler at 50°C for 60 minutes. Once completed, store on ice or at -20°C | ||
+ | |||
− | <li>Once completed, | + | <li>Once completed, use right away or store at -20°C. Thaw chemically-competent cells on ice when ready to use the samples. |
<li>Add 2 µL of the assembled product to the competent cells. Mix gently by pipetting up and down or by flicking the tube 4-5 times. Do not vortex. | <li>Add 2 µL of the assembled product to the competent cells. Mix gently by pipetting up and down or by flicking the tube 4-5 times. Do not vortex. | ||
Line 1,505: | Line 1,144: | ||
</li> | </li> | ||
</ol> | </ol> | ||
− | </li> | + | |
+ | <li>Calculations | ||
+ | <ol> | ||
+ | <li>Example: </br> | ||
+ | Improved frag1=1.01pmoles→Dilute w/10 µL→~100ng/µL and 0.10pmols </br> | ||
+ | Improved frag2=1.40pmoles→Dilute w/10 µL→~100ng/µL and 0.14pmols </br> | ||
+ | Improved frag3=1.64pmoles→Dilute w/10 µL→~100ng/µL and 0.16pmols </br> | ||
+ | pSB1C3 = 25ng/µL → 4µL = 100ng & 0.07 pmol </br> | ||
+ | Total mix = 7µL, 1:1 ratio in ng, 0.47 pmol < 0.50 pmol | ||
+ | </li> | ||
</ol> | </ol> | ||
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− | < | + | </ol> |
− | + | </br> | |
− | + | </br> | |
− | + | </div> | |
− | + | <button class="accordion">Sequencing</button> | |
− | + | <div class="panel-body"> | |
− | + | <ol type="A"> | |
− | + | <li>Materials | |
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− | <li>Materials | + | |
<ol> | <ol> | ||
Line 1,532: | Line 1,171: | ||
<li>Sequencing tube | <li>Sequencing tube | ||
− | <li>2 µM Forward primers | + | <li>2 µM Forward primers |
− | < | + | <ol type="a"> |
<li> | <li> | ||
Line 1,539: | Line 1,178: | ||
(100 M) (x) = (2 M) (50 L) <br/>x = 1 L primer stock | (100 M) (x) = (2 M) (50 L) <br/>x = 1 L primer stock | ||
+ | </li> | ||
</ol> | </ol> | ||
+ | </li> | ||
</ol> | </ol> | ||
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− | |||
<li>Procedure | <li>Procedure | ||
<ol> | <ol> | ||
− | <li>Combine the following components into each tube: 12μL | + | <li>Combine the following components into each tube: 12μL total |
− | < | + | <ol type="a"> |
<li>DNA: (280ng)(1μL/xng) | <li>DNA: (280ng)(1μL/xng) | ||
− | <li>Forward Primer | + | <li>4 µL of 2 µM Forward Primer |
− | <li>Autoclaved Nanowater | + | <li>Autoclaved Nanowater(8µL - volume of DNA) |
− | + | ||
− | + | ||
</li> | </li> | ||
</ol> | </ol> | ||
− | </li> | + | <li>Send tube to Eurofin for sequencing |
+ | </li> | ||
</ol> | </ol> | ||
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</li> | </li> | ||
</ol> | </ol> | ||
− | </li> | + | </li> |
</ol> | </ol> | ||
− | + | </br> | |
− | + | </br> | |
− | </ | + | </div> |
− | + | <button class="accordion">Tryptone Agar (1% tryptone, 0.25% agar)</button> | |
− | + | <div class="panel-body"> | |
− | </ | + | <ol type="A"> |
− | + | <li>Materials | |
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− | <li>Materials | + | |
<ol> | <ol> | ||
− | <li> | + | <li>Autoclaved Nanowater H20 |
<li>Tryptone | <li>Tryptone | ||
Line 1,609: | Line 1,225: | ||
<ol> | <ol> | ||
− | <li>Combine the following components in 500 mL of | + | <li>Combine the following components in 500 mL of autoclaved nanowater H20: |
− | <ol> | + | <ol type="a"> |
<li>5.00 g tryptone | <li>5.00 g tryptone | ||
Line 1,625: | Line 1,241: | ||
</li> | </li> | ||
</ol> | </ol> | ||
− | + | </br> | |
− | + | </br> | |
− | + | </div> | |
− | + | <button class="accordion">Miniprep (QIAprep)</button> | |
− | + | <div class="panel-body"> | |
− | + | <ol type="A"> | |
− | + | <li>Materials | |
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− | <li>Materials | + | |
<ol> | <ol> | ||
Line 1,675: | Line 1,280: | ||
<li>Apply 800 μl supernatant from step 5 to the QIAprep 2.0 spin column by pipetting. For centrifuge processing, follow the instructions marked with a triangle. For vacuum manifold processing, follow the instructions marked with a circle. Centrifuge for 30–60 s and discard the flow-through, or apply vacuum to the manifold to draw the solution through the QIAprep 2.0 spin column and switch off the vacuum source. | <li>Apply 800 μl supernatant from step 5 to the QIAprep 2.0 spin column by pipetting. For centrifuge processing, follow the instructions marked with a triangle. For vacuum manifold processing, follow the instructions marked with a circle. Centrifuge for 30–60 s and discard the flow-through, or apply vacuum to the manifold to draw the solution through the QIAprep 2.0 spin column and switch off the vacuum source. | ||
− | <ol> | + | <ol type="a"> |
<li> Recommended: Wash the QIAprep 2.0 spin column by adding 0.5 ml Buffer PB. Centrifuge for 30–60 s and discard the flow-through, or apply vacuum to the manifold to draw the solution through the QIAprep 2.0 spin column and switch off the vacuum source. | <li> Recommended: Wash the QIAprep 2.0 spin column by adding 0.5 ml Buffer PB. Centrifuge for 30–60 s and discard the flow-through, or apply vacuum to the manifold to draw the solution through the QIAprep 2.0 spin column and switch off the vacuum source. | ||
Line 1,694: | Line 1,299: | ||
</li> | </li> | ||
</ol> | </ol> | ||
− | < | + | </br> |
− | CCMB Solution | + | </br> |
− | </ | + | </div> |
+ | <button class="accordion">CCMB Solution</button> | ||
+ | <div class="panel-body"> | ||
+ | <ol type="A"> | ||
+ | <li>Materials | ||
<ol> | <ol> | ||
− | |||
− | |||
<li> | <li> | ||
Line 1,722: | Line 1,329: | ||
<li> | <li> | ||
Redistilled glycerol- 10 mL per 100mL solution | Redistilled glycerol- 10 mL per 100mL solution | ||
+ | |||
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</li> | </li> | ||
</ol> | </ol> | ||
+ | |||
<li>Procedures | <li>Procedures | ||
<ol> | <ol> | ||
− | + | <li><b>Note: It is absolutely critical to maintain sterile conditions throughout the procedure.</b> | |
<li>Mix the materials together. | <li>Mix the materials together. | ||
− | <ol> | + | <ol type="a"> |
− | <li> | + | <li>Dilute to 100mL solution with Autoclaved nanowater |
</li> | </li> | ||
</ol> | </ol> | ||
Line 1,748: | Line 1,352: | ||
</li> | </li> | ||
</ol> | </ol> | ||
− | + | </br> | |
− | + | </br> | |
− | + | </div> | |
− | + | <button class="accordion">Competent Cell Preparation</button> | |
− | + | <div class="panel-body"> | |
− | + | <ol type="A"> | |
− | + | <li>Materials | |
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− | <li>Materials | + | |
<ol> | <ol> | ||
Line 1,779: | Line 1,371: | ||
<li>Day One | <li>Day One | ||
− | <ol> | + | <ol type="a"> |
<li>Inoculate 5mL of LB with a single colony of the strain of interest. | <li>Inoculate 5mL of LB with a single colony of the strain of interest. | ||
Line 1,788: | Line 1,380: | ||
<li>Day Two | <li>Day Two | ||
− | <ol> | + | <ol type="a"> |
<li>Adjust the temperature of a floor shaker to 30°C. It is <strong>absolutely critical</strong> to maintain the temperature no higher than 30°C. Prior to proceeding to the next step, the chamber temperature should be stabilized for no less than 30 min. | <li>Adjust the temperature of a floor shaker to 30°C. It is <strong>absolutely critical</strong> to maintain the temperature no higher than 30°C. Prior to proceeding to the next step, the chamber temperature should be stabilized for no less than 30 min. | ||
Line 1,829: | Line 1,421: | ||
</li> | </li> | ||
</ol> | </ol> | ||
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− | + | </br> | |
− | + | </div> | |
− | + | <button class="accordion">Motility Characterization</button> | |
− | + | <div class="panel-body"> | |
− | + | <ol type="A"> | |
− | + | <li>Materials | |
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− | <li>Materials | + | |
<ol> | <ol> | ||
Line 1,876: | Line 1,456: | ||
<li>Prepare diffusion plates | <li>Prepare diffusion plates | ||
− | <ol> | + | |
− | + | <ol type="a"> | |
<li>Mark empty plates with a 15 mm line | <li>Mark empty plates with a 15 mm line | ||
Line 1,883: | Line 1,463: | ||
<li>Create approximately 600 mL of tryptone agar for plates | <li>Create approximately 600 mL of tryptone agar for plates | ||
− | <ol> | + | |
− | + | <ol type="I"> | |
<li>All agar should contain 1% tryptone, 0.25% agarose, and 50 µg/mL streptomycin | <li>All agar should contain 1% tryptone, 0.25% agarose, and 50 µg/mL streptomycin | ||
Line 1,894: | Line 1,474: | ||
<li>Pour the following plates: | <li>Pour the following plates: | ||
− | <ol> | + | <ol type="I"> |
<li>6 plates 0% arabinose | <li>6 plates 0% arabinose | ||
Line 1,907: | Line 1,487: | ||
<li>Prepare directionality plates | <li>Prepare directionality plates | ||
− | <ol> | + | <ol type="a"> |
<li>Mark empty plates with a 15 mm line | <li>Mark empty plates with a 15 mm line | ||
Line 1,914: | Line 1,494: | ||
<li>Draw two 15 mm boxes on each end of the 15 mm line | <li>Draw two 15 mm boxes on each end of the 15 mm line | ||
− | <ol> | + | <ol type="I"> |
<li>Label one box “Tryptone” and the other box “Arabinose” | <li>Label one box “Tryptone” and the other box “Arabinose” | ||
Line 1,921: | Line 1,501: | ||
<li>Create tryptone agar for plates | <li>Create tryptone agar for plates | ||
− | <ol> | + | <ol type="I"> |
<li>Agar should contain 1% tryptone, 0.25% agarose, and 50 µg/mL streptomycin | <li>Agar should contain 1% tryptone, 0.25% agarose, and 50 µg/mL streptomycin | ||
Line 1,932: | Line 1,512: | ||
<li>Prepare cells | <li>Prepare cells | ||
− | <ol> | + | <ol type="a"> |
<li>Transform the following DNA constructs into <em>E. coli</em> strain UU2685 (ΔcheZ): | <li>Transform the following DNA constructs into <em>E. coli</em> strain UU2685 (ΔcheZ): | ||
− | <ol> | + | <ol type="I"> |
<li>pBAD + CheZ | <li>pBAD + CheZ | ||
Line 1,944: | Line 1,524: | ||
<li>After transformation, prepare three inoculations each for the following cell types using LB media: | <li>After transformation, prepare three inoculations each for the following cell types using LB media: | ||
− | <ol> | + | <ol type="I"> |
<li>UU2685 from stock (no transformation) | <li>UU2685 from stock (no transformation) | ||
Line 1,965: | Line 1,545: | ||
<li>Diffusion assay | <li>Diffusion assay | ||
− | <ol> | + | <ol type="a"> |
<li>Plate 5 µL of UU2685 onto a 0% arabinose plate. Place the cells on the dot labeled in the middle of the plate. | <li>Plate 5 µL of UU2685 onto a 0% arabinose plate. Place the cells on the dot labeled in the middle of the plate. | ||
Line 1,974: | Line 1,554: | ||
<li>Repeat steps 1-3 for all cell cultures to create the following plates: | <li>Repeat steps 1-3 for all cell cultures to create the following plates: | ||
− | <ol> | + | <ol type="I"> |
<li>UU2685 | <li>UU2685 | ||
− | <ol> | + | <ol type="i"> |
<li>Three 0% arabinose plates | <li>Three 0% arabinose plates | ||
Line 1,988: | Line 1,568: | ||
<li>pBAD + CheZ | <li>pBAD + CheZ | ||
− | <ol> | + | <ol type="i"> |
<li>Three 0% arabinose plates | <li>Three 0% arabinose plates | ||
Line 1,999: | Line 1,579: | ||
<li>pBAD + CheZ + YbaQ | <li>pBAD + CheZ + YbaQ | ||
− | <ol> | + | <ol type="i"> |
<li>Three 0% arabinose plates | <li>Three 0% arabinose plates | ||
Line 2,014: | Line 1,594: | ||
<li>Directionality | <li>Directionality | ||
− | <ol> | + | <ol type="a"> |
<li>Plate 5 µL of UU2685 onto the dot in the center of the plate | <li>Plate 5 µL of UU2685 onto the dot in the center of the plate | ||
Line 2,027: | Line 1,607: | ||
<li>Repeat steps 1-5 for all cell cultures to create the following plates: | <li>Repeat steps 1-5 for all cell cultures to create the following plates: | ||
− | <ol> | + | <ol type="I"> |
<li>UU2685 | <li>UU2685 | ||
− | <ol> | + | <ol type="i"> |
<li>Three 0% arabinose plates | <li>Three 0% arabinose plates | ||
Line 2,041: | Line 1,621: | ||
<li>pBAD + CheZ | <li>pBAD + CheZ | ||
− | <ol> | + | <ol type="i"> |
<li>Three 0% arabinose plates | <li>Three 0% arabinose plates | ||
Line 2,052: | Line 1,632: | ||
<li>pBAD + CheZ + YbaQ | <li>pBAD + CheZ + YbaQ | ||
− | <ol> | + | <ol type="i"> |
<li>Three 0% arabinose plates | <li>Three 0% arabinose plates | ||
Line 2,067: | Line 1,647: | ||
<li>The next day | <li>The next day | ||
− | <ol> | + | <ol type="a"> |
<li>Use the 15 mm lines marked on the plates to determine the radius of growth | <li>Use the 15 mm lines marked on the plates to determine the radius of growth | ||
Line 2,076: | Line 1,656: | ||
</li> | </li> | ||
</ol> | </ol> | ||
− | + | </br> | |
− | + | </br> | |
− | + | </div> | |
− | + | <button class="accordion">Sensing Characterization</button> | |
− | + | <div class="panel-body"> | |
− | + | <ol type="A"> | |
− | + | <li>Materials | |
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<ol> | <ol> | ||
Line 2,130: | Line 1,699: | ||
<li>Perform a dilution for each sample into 13 individual tubes (triplicates of 4 conditions per sample and 1 tube to monitor OD growth) with more LB to OD<sub>600</sub> 0.05 and let culture until 0.1. The new volume should be 1 mL. There should be 52 tubes total. Antibiotic should be added also. | <li>Perform a dilution for each sample into 13 individual tubes (triplicates of 4 conditions per sample and 1 tube to monitor OD growth) with more LB to OD<sub>600</sub> 0.05 and let culture until 0.1. The new volume should be 1 mL. There should be 52 tubes total. Antibiotic should be added also. | ||
− | <ol> | + | <ol type="a"> |
<li>(OD1)(x)=(0.05)(1mL) | <li>(OD1)(x)=(0.05)(1mL) | ||
Line 2,137: | Line 1,706: | ||
x = volume from culture | x = volume from culture | ||
</p> | </p> | ||
− | < | + | </li> |
− | + | </ol> | |
<li>After OD<sub>600</sub> is 0.1, add the correct amounts of AIP and 20% L-arabinose for each condition: | <li>After OD<sub>600</sub> is 0.1, add the correct amounts of AIP and 20% L-arabinose for each condition: | ||
− | <ol> | + | <ol type="a"> |
<li>AIP & arabinose: | <li>AIP & arabinose: | ||
− | <ol> | + | <ol type="I"> |
<li>AIP: 4 µL | <li>AIP: 4 µL | ||
Line 2,152: | Line 1,721: | ||
<li>AIP: | <li>AIP: | ||
− | <ol> | + | <ol type="I"> |
<li>AIP: 4 µL | <li>AIP: 4 µL | ||
Line 2,161: | Line 1,730: | ||
<li>arabinose: | <li>arabinose: | ||
− | <ol> | + | <ol type="I"> |
<li>AIP: 0 µL | <li>AIP: 0 µL | ||
Line 2,170: | Line 1,739: | ||
<li>Neither: | <li>Neither: | ||
− | <ol> | + | <ol type="I"> |
<li>AIP: 0 µL | <li>AIP: 0 µL | ||
Line 2,185: | Line 1,754: | ||
<li>Wash and resuspend the hour 0 cells in PBS for initial measurement | <li>Wash and resuspend the hour 0 cells in PBS for initial measurement | ||
− | <ol> | + | <ol type="a"> |
<li>Centrifuge at 5,000 g for 10 minutes | <li>Centrifuge at 5,000 g for 10 minutes | ||
− | <ol> | + | <ol type="I"> |
<li>Cool centrifuge to 4 °C | <li>Cool centrifuge to 4 °C | ||
Line 2,205: | Line 1,774: | ||
<li>Remove 300 µL from each culture after 8 hours and wash and resuspend with PBS | <li>Remove 300 µL from each culture after 8 hours and wash and resuspend with PBS | ||
− | <ol> | + | <ol type="a"> |
<li>Centrifuge at 5000 g for 10 minutes | <li>Centrifuge at 5000 g for 10 minutes | ||
− | <ol> | + | <ol type="I"> |
<li>Cool centrifuge to 4 °C | <li>Cool centrifuge to 4 °C | ||
Line 2,225: | Line 1,794: | ||
<li>Save the rest of the cultures for flow cytometry | <li>Save the rest of the cultures for flow cytometry | ||
− | <ol> | + | <ol type="a"> |
<li>Place samples in 4 °C refrigerator to halt growth | <li>Place samples in 4 °C refrigerator to halt growth | ||
Line 2,239: | Line 1,808: | ||
<li>AIP: | <li>AIP: | ||
− | <ol> | + | <ol type="a"> |
<li>(2500 µM)(v) = (10 µM)(1000 µL) v = 4 µL | <li>(2500 µM)(v) = (10 µM)(1000 µL) v = 4 µL | ||
Line 2,246: | Line 1,815: | ||
<li>Arabinose: | <li>Arabinose: | ||
− | <ol> | + | <ol type="a"> |
<li>(20%)(v) = (0.2%)(1000 µL) v = 10 µL | <li>(20%)(v) = (0.2%)(1000 µL) v = 10 µL | ||
Line 2,259: | Line 1,828: | ||
</li> | </li> | ||
</ol> | </ol> | ||
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− | + | </div> | |
− | + | <button class="accordion">Killing Characterization</button> | |
− | + | <div class="panel-body"> | |
− | + | <ol type="A"> | |
− | + | <li>Materials | |
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− | <li>Materials | + | |
<ol> | <ol> | ||
Line 2,320: | Line 1,877: | ||
<li>Grow to an OD equal to or greater than 2.0 | <li>Grow to an OD equal to or greater than 2.0 | ||
− | < | + | <ol type="a"> |
+ | <li>Check OD with a spectrophotometer using a 1 mL M9 broth as the blank and a 1:10 dilution of culture. Dilute using M9 broth.</li> | ||
+ | </ol> | ||
− | |||
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− | |||
− | |||
<li>Place the entire culture into a 1 L flask containing 250 mL of M9 broth | <li>Place the entire culture into a 1 L flask containing 250 mL of M9 broth | ||
Line 2,373: | Line 1,928: | ||
</li> | </li> | ||
</ol> | </ol> | ||
− | </ | + | </br> |
− | </ | + | </br> |
− | + | </div> | |
− | + | <button class="accordion">Bradford Assay</button> | |
− | + | <div class="panel-body"> | |
− | + | <ol type="A"> | |
− | + | <li>Materials | |
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<ol> | <ol> | ||
Line 2,409: | Line 1,949: | ||
<li>Prepare the following standard solutions with bovine serum albumin (BSA) and autoclaved nanowater: | <li>Prepare the following standard solutions with bovine serum albumin (BSA) and autoclaved nanowater: | ||
− | <ol> | + | <ol type="a"> |
<li>0 mg/mL BSA | <li>0 mg/mL BSA | ||
Line 2,426: | Line 1,966: | ||
<li>Dilute the protein sample with autoclaved nanowater to create the following dilutions: | <li>Dilute the protein sample with autoclaved nanowater to create the following dilutions: | ||
− | <ol> | + | <ol type="a"> |
<li>1:30 dilution | <li>1:30 dilution | ||
Line 2,439: | Line 1,979: | ||
<li>Prepare a sample for the assay by adding 5 µL of the desired sample to 250 µL of Coomassie Blue dye. Pipet to mix. | <li>Prepare a sample for the assay by adding 5 µL of the desired sample to 250 µL of Coomassie Blue dye. Pipet to mix. | ||
− | <ol> | + | <ol type="a"> |
<li>Create three samples for each standard solution and 3 samples for each protein dilution to allow triplicate measurements | <li>Create three samples for each standard solution and 3 samples for each protein dilution to allow triplicate measurements | ||
Line 2,458: | Line 1,998: | ||
</li> | </li> | ||
</ol> | </ol> | ||
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− | + | <button class="accordion">20% L-arabinose Preparation</button> | |
− | + | <div class="panel-body"> | |
− | + | <ol type="A"> | |
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<ol> | <ol> | ||
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</li> | </li> | ||
</ol> | </ol> | ||
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− | + | <button class="accordion">M9 Salts Preparation (500 mL)</button> | |
− | + | <div class="panel-body"> | |
− | + | <ol type="A"> | |
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<ol> | <ol> | ||
+ | <li>Na<sub>2</sub>HPO<sub>4</sub>-7H<sub>2</sub>O | ||
+ | <li>KH<sub>2</sub>PO<sub>4</sub> | ||
+ | <li>NaCl | ||
+ | <li>NH<sub>4</sub>Cl | ||
+ | <li>Autoclaved Nanowater | ||
+ | </li> | ||
+ | </ol> | ||
− | <li> | + | <li> Procedure |
<ol> | <ol> | ||
− | + | <li> Add the following quantities into a 1 L bottle: | |
+ | <ol type="a"> | ||
<li>32g Na<sub>2</sub>HPO<sub>4</sub>-7H<sub>2</sub>O | <li>32g Na<sub>2</sub>HPO<sub>4</sub>-7H<sub>2</sub>O | ||
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<li>500 mL nanowater | <li>500 mL nanowater | ||
− | + | </li> | |
− | <li>Autoclave | + | </ol> |
+ | <li>Autoclave the solution | ||
</li> | </li> | ||
</ol> | </ol> | ||
</li> | </li> | ||
</ol> | </ol> | ||
− | < | + | </br> |
− | M9 Broth ( | + | </br> |
− | </ | + | </div> |
+ | <button class="accordion">M9 Broth Preparation (1 L)</button> | ||
+ | <div class="panel-body"> | ||
+ | <ol type="A"> | ||
+ | <li>Materials | ||
<ol> | <ol> | ||
+ | <li>1M MgSO4 solution | ||
+ | <li>1M CaCl2 solution | ||
+ | <li>glycerol | ||
+ | <li>M9 salt solution | ||
+ | <li>0.3M Leucine (in 1M HCl solution) (filter sterilized) | ||
+ | </li> | ||
+ | </ol> | ||
− | <li> | + | <li>Procedure |
<ol> | <ol> | ||
+ | <li>Autoclave the MgSO4, CaCl2, and M9 salt solutions | ||
+ | <li>Add the following quantities together: | ||
+ | <ol type="a"> | ||
+ | <li>2 mL of autoclaved 1M MgSO4 solution | ||
− | <li> | + | <li>100µL of autoclaved 1M CaCl2 solution |
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<li>20 mL of glycerol | <li>20 mL of glycerol | ||
− | <li>200 mL of | + | <li>200 mL of autoclaved M9 salt solution |
<li>0.5 mL of 0.3M Leucine (in 1M HCl solution) (filter sterilized) | <li>0.5 mL of 0.3M Leucine (in 1M HCl solution) (filter sterilized) | ||
</li> | </li> | ||
</ol> | </ol> | ||
+ | </li> | ||
+ | </ol> | ||
+ | |||
</li> | </li> | ||
</ol> | </ol> | ||
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− | + | </div> | |
− | + | <button class="accordion">Ligation Cloning</button> | |
− | + | <div class="panel-body"> | |
− | + | <ol type="A"> | |
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<ol> | <ol> | ||
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</li> | </li> | ||
</ol> | </ol> | ||
− | < | + | <li>Procedure |
− | Procedure | + | |
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<ol> | <ol> | ||
− | + | <li> Look for the insert size removed from the plasmid and subtract it from the plasmid size to determine the new plasmid size after digestion | |
+ | <ol type="a"> | ||
+ | <li>Insert construct size: _____________bp | ||
+ | <li>Plasmid size: ____________ bp – insert size=______________ bp | ||
+ | </li> | ||
+ | </ol> | ||
+ | <li>Determine the insert 3:1 ratio using the following formula: | ||
+ | <ol type="a"> | ||
+ | <li><sup>insert size bp</sup>⁄<sub>digested plasmid bp</sub> * 3 = insert bp 3 : 1 ratio * 1 μL= x μL | ||
+ | </li> | ||
+ | </ol> | ||
+ | <li>Using the results from the mentioned formula determine the nanograms needed for a concentration of 50 ng µL<sup>-1</sup> | ||
+ | <ol type="a"> | ||
+ | <li><sup>x μL</sup>⁄<sub>1</sub> * <sup>50 ng</sup>⁄<sub>1μL</sub> = x ng | ||
+ | </li> | ||
+ | </ol> | ||
+ | <li>Determine the volume needed from the construct (insert) using the following formula: | ||
+ | <ol type="a"> | ||
+ | <li><sup>x μL</sup>⁄<sub>1</sub> * <sup>1 μL</sup>⁄<sub>concentration of ng µL<sup>-1</sup></sub> = x μL of construct needed | ||
+ | </li> | ||
+ | </ol> | ||
+ | <li>determine the volume needed from the plasmid (vector) using the following formula: | ||
+ | <ol type="a"> | ||
+ | <li><sup>50 μL</sup>⁄<sub>1</sub> * <sup>1 μL</sup>⁄<sub>plasmid concentration ( ng µL<sup>-1</sup>)</sub> = x μL of plasmid needed | ||
+ | </li> | ||
+ | </ol> | ||
+ | <li>Prepare the following mix for reaction for 10μL | ||
+ | <ol type="a"> | ||
<li>T4 buffer 1μL | <li>T4 buffer 1μL | ||
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</li> | </li> | ||
</ol> | </ol> | ||
− | + | </li> | |
− | + | </ol> | |
− | + | </li> | |
− | + | </ol> | |
− | + | </br> | |
− | + | </br> | |
+ | </div> | ||
+ | |||
+ | </br> | ||
+ | </br> | ||
</div> <!-- end of page content --> | </div> <!-- end of page content --> | ||
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</div><!-- end of container --> | </div><!-- end of container --> | ||
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<div class="footer"> | <div class="footer"> | ||
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</body> | </body> | ||
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+ | panel.style.display = "none"; | ||
+ | } else { | ||
+ | panel.style.display = "block"; | ||
+ | } | ||
}); | }); | ||
− | + | } | |
+ | </script> | ||
</html> | </html> |
Latest revision as of 15:02, 16 December 2019