Team:UT Dallas/Protocols

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      <h1><span>UTDallas</span> iGem<small></small></h1>
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          <h2><span> <b>Protocols</b></span></h2>
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          <h2><span></span>Ligation Protocol</h2>
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          <div class="clr"></div>
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          <p><li type = "disk">Determine insert to vector ratios<li type = "disk">Calculate the amount of insert needed if 50ng of vector is used (can use different amount of vector)<li type = "disk">In a PCR tube add the following:<blockquote><li type = "circle">50ng of vector<li type = "circle">Amount of insert based on ratios (calculated in second step)<li type = "circle">2uL of buffer<li type = "circle">2uL of DNA ligase<li type = "circle">Amount of water to bring total volume to 20uL</blockquote><li type = "disk">Incubate overnight at 14 degrees Celsius</p></li><p>Note: We used T4 DNA ligase and buffer from NEB</p>
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          <h2><span></span>Gel Purification Protocol (QIAquick Gel Extraction Kit)</h2>
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          <div class="clr"></div>
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          <p><li type = "disk">Excise DNA fragment from the agarose gel with a clean, sharp scalpel<li type = "disk">Weigh the gel slice in a microcentrifuge tube.<li type = "disk">Add 3 volumes of Buffer QG to 1 volume of gel (100mg~100uL)<li type = "disk">Incubate at 50 degrees Celsius for 10 min (until the gel slice has completely dissolved)<li type = "disk">After the gel slice has dissolved completely, check that the color of the mixture is yellow<li type = "disk">Apply the sample to a QIAquick column, and centrifuge for 1 min<li type = "circle"><blockquote>Maximum volume of the column is 800uL. For samples larger than this, simply load and spin again.</blockquote><li type = "disk">Discard flow-through and place QIAquick column back in the same collection tube<li type = "disk">To wash, add 750uL of Buffer PE to column and centrifuge for 1 min.<li type = "disk">Discard the flow-through and centrifuge for additional 1 min. at 13,000rpm<li type = "disk">Place QIAquick column into a clean 1.5 mL microcentrifuge tube<li type = "disk">To elute DNA, add 50uL of Buffer EB to the center of the QIAquick membrane, let the column stand for 1 min. and then centrifuge the column for 1 min.</p></li>
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          <h2><span></span>Gel Electrophoresis Protocol</h2>
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          <div class="clr"></div>
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          <p><li type = "disk">Making a 1% agarose gel<blockquote><li type = "circle">100mL 1X TBE buffer<li type = "circle">1g agarose<li type = "circle">microwave until agarose dissolves<li type = "circle">let mixture cool<li type = "circle">when cool add 8-10uL ethidium bromide<li type = "circle">stir gently, let cool<li type = "circle">pour into plate with comb already in place<li type = "circle">let harden</blockquote><li type = "disk">Using the gel<blockquote><li type = "circle">Add loading buffer to DNA (for 100uL DNA, add 20uL loading buffer)<li type = "circle">Load 2uL of DNA ladder into the gel<li type = "circle">Load DNA into the gel<li type = "circle">Run at 130V for 30min-1hr</p></li></blockquote>
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          <h2><span></span>Digestion Protocol</h2>
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          <div class="clr"></div>
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          <p><li type = "disk">Using a microcentrifuge tube add the following:<blockquote><li type = "circle">~3000-5000 ng of DNA<li type = "circle">10uL Buffer 4<li type = "circle">10uL BSA<li type = "circle">5uL of appropriate enzyme (if doing a double digest, use 5 uL of both enzymes)<li type = "circle">Amount of H2O needed to make final volume 100uL</blockquote><li type = "disk">Incubate at 37 degrees Celsius for 1hr and 30min</p></li><p>Note: We used the following enzymes from NEB: EcoRI-HF, PstI-HF, SpeI, and XbaI. All of which can be double digested with each other using Buffer 4.</p>
 +
          <h2><span></span>Preparing LB+Appropriate Antibiotic Protocol</h2>
 +
          <div class="clr"></div>
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          <p><li type = "disk">200 mL LB broth<li type = "disk">Autoclave<blockquote><li type = "circle">Put control thermometer in H2O (from the sink)<li type = "circle">Select vented container mode (Do Not Change Program)</blockquote><li type = "disk">Let cool to 50 degrees Celsius<li type = "disk">Add antibiotic (50-100 ug/mL) (10 mg total)<blockquote><li type = "circle">Weigh on paper<li type = "circle">Add to 0.5 mL DI H2O<li type = "circle">Add to LB mixture when cool enough</blockquote><li type = "disk">Store at 4 degrees Celsius</p></li>
 +
          <h2><span></span>Preparing Agar Plates Protocol (Makes 12 (15mm) Plates)
 +
</h2>
 +
          <div class="clr"></div>
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          <p><li type = "disk">300 mL DI H2O + 11 g LB agar<li type = "disk">Autoclave<blockquote><li type = "circle">Put control thermometer in H2O (from the sink)<li type = "circle">Select vented container mode (Do Not Change Program)</blockquote><li type = "disk">Mix well after autoclaving; let cool to 50 degrees Celsius<li type = "disk">Add antibiotic (50 to 100 µg/mL) (15 mg total)<blockquote><li type = "circle">Weigh on paper<li type = "circle">Add to 0.5 mL DI H2O<li type = "circle">Add to LB mixture when cool enough</blockquote><li type = "disk">Plate<blockquote><li type = "circle">Under flame open lids of all plates<li type = "circle">Slowly pour agar into plate, avoiding bubbles, when it touches all edges stop pouring<li type = "circle">Let sit under flames until gel solidifies<li type = "circle">Replace lids on plates</blockquote><li type = "disk">Store upside down at 4 degrees Celsius</p></li>
 +
          <h2><span></span>Preparing Competent Cells Protocol</h2>
 +
          <div class="clr"></div>
 +
          <p><li type = "disk">Place 1 colony in 5 mL of LB (with antibiotics if appropriate) Grow overnight at 37 degrees Celsius and 200-300 rpm<li type = "disk">Inoculate 0.25 mL of the overnight strain into 25 mL of LB<li type = "disk">Shake at 37oC until the OD650 is 0.6-0.7<li type = "disk">Harvest cells and resuspend in 12.5 mL ice cold 0.1M MgCl2<li type = "disk">Harvest immediately and resuspend in 7.5 mL cold 0.1M CaCl2<li type = "disk">Leave on ice for 30 minutes. Harvest and resuspend in 2.5 mL cold 0.1M CaCl2<li type = "disk">Leave on ice for 30 minutes<li type = "disk">For long term storage, use 0.1M CaCl2 in 15% glycerol at step 6 and store cells at -800 degrees Celsius</p></li><p>
 +
Note: Harvest cells at 5000 rpm for 10 minutes at 4 degrees Celsius</p>
 +
          <h2><span></span>Miniprep Protocol (from QIAprep Spin Miniprep Kit)</h2>
 +
          <div class="clr"></div>
 +
          <p><li type = "disk">Harvest cells at 5400g 10 minutes 40 degrees Celsius (possibly program 1)<li type = "disk">Resuspend pelleted bacterial cells in 250 µL Buffer P1 and transfer to a microcentrifuge tube<li type = "disk">Add 250 µL Buffer P2 and mix thoroughly by inverting the tube 4-6 times<li type = "disk">Add 350 µL Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times<li type = "disk">Centrifuge for 10 minutes at 13000 rpm (~17900g) in a table-top microcentrifuge<li type = "disk">Apply the supernatant (from step 4) to the QIA prep spin column by decanting or pipetting<li type = "disk">Centrifuge for 30-60 seconds. Discard the flow-through<li type = "disk">Wash QIA prep spin column by adding 0.75 mL Buffer PE and centrifuging for 30-60 seconds<li type = "disk">Discard the flow-through, and centrifuge for 1 minute to remove residual wash buffer<li type = "disk">To elute DNA, place the QIA prep column in a clean 1.5 mL microcentrifuge tube. Add 50 µL Buffer EB or water to the center of each QIA prep spin column, let stand for 1 minute and centrifuge for 1 minute.
 +
</p></li>
 +
          <h2><span></span>Preparing Glycerol Stock Protocol</h2>
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          <p><li type = "disk">Add 150 µL of 50% glycerol to 350 µL of cells<li type = "disk">Place in -80oC freezer</p></li>
 +
          <h2><span></span>Transformation Protocol</h2>
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          <div class="clr"></div>
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          <p><li type = "disk">With a pipette tip, punch a hole through the foil cover of the DNA plate<li type = "disk">Add 10 µL of DI water<li type = "disk">Thaw competent cells on ice<li type = "disk">Add 1-2 µL of resuspend DNA and 50 µL of thawed competent cells to labeled tubes<li type = "disk">Incubate the cells on ice for 30 minutes<li type = "disk">Heat shock the cells at 42 degrees Celsius for 45 sec<li type = "disk">Incubate the cells on ice for 2 minutes<li type = "disk">Under flame, add 450 µL SOC broth<li type = "disk">Incubate at 37 degrees Celsius for 1 hour while rotating or shaking at 300rpm<li type = "disk">Spread cells on appropriate antibiotic LB plates (usually 100 µL)<li type = "disk">Incubate at 37 degrees Celsius for 18-24 hours<li type = "disk">Take a colony, put in 3 mL of LB + appropriate antibiotic<li type = "disk">Use resulting culture to miniprep DNA and make your own glycerol stock</p></li>
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Latest revision as of 02:11, 3 September 2011

biz solution

Protocols

Ligation Protocol

  • Determine insert to vector ratios
  • Calculate the amount of insert needed if 50ng of vector is used (can use different amount of vector)
  • In a PCR tube add the following:
  • 50ng of vector
  • Amount of insert based on ratios (calculated in second step)
  • 2uL of buffer
  • 2uL of DNA ligase
  • Amount of water to bring total volume to 20uL
  • Incubate overnight at 14 degrees Celsius

    • Note: We used T4 DNA ligase and buffer from NEB

    Gel Purification Protocol (QIAquick Gel Extraction Kit)

  • Excise DNA fragment from the agarose gel with a clean, sharp scalpel
  • Weigh the gel slice in a microcentrifuge tube.
  • Add 3 volumes of Buffer QG to 1 volume of gel (100mg~100uL)
  • Incubate at 50 degrees Celsius for 10 min (until the gel slice has completely dissolved)
  • After the gel slice has dissolved completely, check that the color of the mixture is yellow
  • Apply the sample to a QIAquick column, and centrifuge for 1 min
  • Maximum volume of the column is 800uL. For samples larger than this, simply load and spin again.
  • Discard flow-through and place QIAquick column back in the same collection tube
  • To wash, add 750uL of Buffer PE to column and centrifuge for 1 min.
  • Discard the flow-through and centrifuge for additional 1 min. at 13,000rpm
  • Place QIAquick column into a clean 1.5 mL microcentrifuge tube
  • To elute DNA, add 50uL of Buffer EB to the center of the QIAquick membrane, let the column stand for 1 min. and then centrifuge the column for 1 min.

    • Gel Electrophoresis Protocol

  • Making a 1% agarose gel
  • 100mL 1X TBE buffer
  • 1g agarose
  • microwave until agarose dissolves
  • let mixture cool
  • when cool add 8-10uL ethidium bromide
  • stir gently, let cool
  • pour into plate with comb already in place
  • let harden
  • Using the gel
  • Add loading buffer to DNA (for 100uL DNA, add 20uL loading buffer)
  • Load 2uL of DNA ladder into the gel
  • Load DNA into the gel
  • Run at 130V for 30min-1hr

    • Digestion Protocol

  • Using a microcentrifuge tube add the following:
  • ~3000-5000 ng of DNA
  • 10uL Buffer 4
  • 10uL BSA
  • 5uL of appropriate enzyme (if doing a double digest, use 5 uL of both enzymes)
  • Amount of H2O needed to make final volume 100uL
  • Incubate at 37 degrees Celsius for 1hr and 30min

    • Note: We used the following enzymes from NEB: EcoRI-HF, PstI-HF, SpeI, and XbaI. All of which can be double digested with each other using Buffer 4.

    Preparing LB+Appropriate Antibiotic Protocol

  • 200 mL LB broth
  • Autoclave
  • Put control thermometer in H2O (from the sink)
  • Select vented container mode (Do Not Change Program)
  • Let cool to 50 degrees Celsius
  • Add antibiotic (50-100 ug/mL) (10 mg total)
  • Weigh on paper
  • Add to 0.5 mL DI H2O
  • Add to LB mixture when cool enough
  • Store at 4 degrees Celsius

    • Preparing Agar Plates Protocol (Makes 12 (15mm) Plates)

  • 300 mL DI H2O + 11 g LB agar
  • Autoclave
  • Put control thermometer in H2O (from the sink)
  • Select vented container mode (Do Not Change Program)
  • Mix well after autoclaving; let cool to 50 degrees Celsius
  • Add antibiotic (50 to 100 µg/mL) (15 mg total)
  • Weigh on paper
  • Add to 0.5 mL DI H2O
  • Add to LB mixture when cool enough
  • Plate
  • Under flame open lids of all plates
  • Slowly pour agar into plate, avoiding bubbles, when it touches all edges stop pouring
  • Let sit under flames until gel solidifies
  • Replace lids on plates
  • Store upside down at 4 degrees Celsius

    • Preparing Competent Cells Protocol

  • Place 1 colony in 5 mL of LB (with antibiotics if appropriate) Grow overnight at 37 degrees Celsius and 200-300 rpm
  • Inoculate 0.25 mL of the overnight strain into 25 mL of LB
  • Shake at 37oC until the OD650 is 0.6-0.7
  • Harvest cells and resuspend in 12.5 mL ice cold 0.1M MgCl2
  • Harvest immediately and resuspend in 7.5 mL cold 0.1M CaCl2
  • Leave on ice for 30 minutes. Harvest and resuspend in 2.5 mL cold 0.1M CaCl2
  • Leave on ice for 30 minutes
  • For long term storage, use 0.1M CaCl2 in 15% glycerol at step 6 and store cells at -800 degrees Celsius

    • Note: Harvest cells at 5000 rpm for 10 minutes at 4 degrees Celsius

    Miniprep Protocol (from QIAprep Spin Miniprep Kit)

  • Harvest cells at 5400g 10 minutes 40 degrees Celsius (possibly program 1)
  • Resuspend pelleted bacterial cells in 250 µL Buffer P1 and transfer to a microcentrifuge tube
  • Add 250 µL Buffer P2 and mix thoroughly by inverting the tube 4-6 times
  • Add 350 µL Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times
  • Centrifuge for 10 minutes at 13000 rpm (~17900g) in a table-top microcentrifuge
  • Apply the supernatant (from step 4) to the QIA prep spin column by decanting or pipetting
  • Centrifuge for 30-60 seconds. Discard the flow-through
  • Wash QIA prep spin column by adding 0.75 mL Buffer PE and centrifuging for 30-60 seconds
  • Discard the flow-through, and centrifuge for 1 minute to remove residual wash buffer
  • To elute DNA, place the QIA prep column in a clean 1.5 mL microcentrifuge tube. Add 50 µL Buffer EB or water to the center of each QIA prep spin column, let stand for 1 minute and centrifuge for 1 minute.

    • Preparing Glycerol Stock Protocol

  • Add 150 µL of 50% glycerol to 350 µL of cells
  • Place in -80oC freezer

    • Transformation Protocol

  • With a pipette tip, punch a hole through the foil cover of the DNA plate
  • Add 10 µL of DI water
  • Thaw competent cells on ice
  • Add 1-2 µL of resuspend DNA and 50 µL of thawed competent cells to labeled tubes
  • Incubate the cells on ice for 30 minutes
  • Heat shock the cells at 42 degrees Celsius for 45 sec
  • Incubate the cells on ice for 2 minutes
  • Under flame, add 450 µL SOC broth
  • Incubate at 37 degrees Celsius for 1 hour while rotating or shaking at 300rpm
  • Spread cells on appropriate antibiotic LB plates (usually 100 µL)
  • Incubate at 37 degrees Celsius for 18-24 hours
  • Take a colony, put in 3 mL of LB + appropriate antibiotic
  • Use resulting culture to miniprep DNA and make your own glycerol stock

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    UT Dallas

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    Notebook

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    Retrieved from "http://2011.igem.org/Team:UT_Dallas/Protocols"