Difference between revisions of "Team:UIUC Illinois/Experiments"

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{{UIUC_Illinois}}
 
{{UIUC_Illinois}}
 
<html>
 
<html>
 +
<h2>A. Using a Lab Notebook</h2>
  
−
<h2>Experiments &amp; Protocols</h2>
+
<ol>
 +
    <li>Always include the date and your initials or signature.</li>
 +
    <li>Procedures from previous labs do not have to be rewritten, but should be clearly referenced.</li>
 +
    <li>Always Include:<ul>
 +
    <li>Any and all calculations</li>
 +
    <li>Number of cells seeded</li>
 +
    <li>Components of solutions</li>
 +
    <li>Type of cells</li>
 +
    <li>Type of media</li>
 +
    <li>Brand names of solutions used (Invitrogen, Bio-Rad, Promega, etc.)</li>
 +
    <li>Observations such as confluency, color of media, etc.</li></ul></li>
 +
    <li>Always write directly in the notebook, rather than writing it on a separate sheet and copying it later.</li>
 +
    <li>Do no leave large open spaces. If you do, cross these out.</li>
 +
    <li>If you cross out text, make sure it is still legible underneath.</li>
 +
    <li>Number all pages in the notebook, and do not tear pages out.</li>
 +
    <li>Write all entries in ink.</li>
 +
    <li>Use original documentation when possible (e.g. machine printout rather than copying data by hand.)</li>
 +
    <li>Be thorough! Somebody should be able to repeat your experiments by looking at the lab notebook.</li>
 +
</ol>
  
−
<p>Describe the experiments, research and protocols you used in your iGEM project.</p>
+
<h2>Making LB</h2>
 +
<ol>
 +
    <li>Add 20 g of LB powder to 1 L of water and mix.</li>
 +
    <li>Autoclave on a liquid cycle.</li>
 +
    <li>If adding antibiotics, wait until the LB has cooled enough to comfortably touch the bottle. Otherwise, the heat will start to break them down.</li>
 +
</ol>
  
−
<h5>What should this page contain?</h5>
+
<h2>Pouring plates</h2>
−
<ul>
+
<ol>
−
<li> Protocols </li>
+
    <li>Add 20 g of LB powder and 14 g of agar (NOT agarose) to 1 L of water and mix.</li>
−
<li> Experiments </li>
+
    <li>Autoclave on a liquid cycle</li>
−
<li>Documentation of the development of your project </li>
+
    <li>Wait until the LB agar has cooled enough to comfortably touch the bottle.</li>
−
</ul>
+
    <li>If desired, add antibiotics now.</li>
−
 
+
    <li>Pour 10 - 20 mL of LB agar into each plate. This can be done by eye, but using a pipet-aid will give more consistent results</li>
−
 
+
    <li>If any bubbles formed on the surface of your plates, quickly pop them using a pipet tip.</li>
−
 
+
    <li>Allow the plates to solidify.</li>
−
<h4>Inspiration</h4>
+
    <li>Wrap plates in a bag, sleeve, or Parafilm.</li>
−
<ul>
+
    <li>Store upside down at 4 degrees.</li>
−
<li><a href="https://2014.igem.org/Team:Colombia/Protocols">2014 Colombia </a></li>
+
</ol>
−
<li><a href="https://2014.igem.org/Team:Imperial/Protocols">2014 Imperial </a></li>
+
<h2>Transformation</h2>
−
<li><a href="https://2014.igem.org/Team:Caltech/Project/Experiments">2014 Caltech </a></li>
+
<ol>
−
</ul>
+
    <li>Take competent cells out of freezer and thaw on ice for 20-30 minutes. Take only as many as you need; they lose competency if re-frozen.</li>
−
</div>
+
    <li>Take agar plates out of storage and allow them to warm up to room temperature.</li>
 +
    <li>Add 10 pg to 100 ng of DNA (usually about 1-5 uL depending on concentration) to 20 - 50 uL of competent cells.</li>
 +
    <li>Mix tubes by gently flicking.</li>
 +
    <li>Place tubes back on ice for 20-30 minutes.</li>
 +
    <li>Heat shock transformation by placing it in 42 degree C water bath for 45 seconds.</li>
 +
    Note: Some strains of e coli may require shorter or longer heat shock times. Always check the strain before transforming!</li>
 +
    <li>Set tubes back on ice for 2 minutes.</li>
 +
    <li>Add 250 - 500 uL of LB and grow in the 37 shaking incubator for 45 minutes. If you are plating on ampicillin, this step can be skipped.</li>
 +
    <li>Plate 50 uL of cells on each plate using beads or cell spreader.</li>
 +
    <li>Grow overnight at 37 degrees C.</li>
 +
</ol>
 +
<h2>Making electrophoresis gels</h2>
 +
<ol>
 +
    <li>Add 1 g of agarose to 100 mL of 10x TAE buffer.</li>
 +
    <li>Heat in the microwave for about 1 minute and 45 seconds, until the solution has come to a boil.</li>
 +
    <li>Allow the liquid to cool for 5 minutes.</li>
 +
    <li>Add 10 uL of Midori Green and swirl gently to mix.</li>
 +
    <li>Pour 30 - 50 mL into gel tray with comb. Use as little gel as possible if you plan to extract and purify plasmids from it later.</li></li>
 +
    <li>Let sit about 30 minutes to cast.</li>
 +
    <li>Note: this makes a 1% gel, which works for most purposes. However, a lower percentage, such as 0.7% gel, is best for gel extractions, and as high as 2% agarose can be used when resolving large bands of DNA.</li>
 +
</ol>
 +
<h2>Gel Electrophoresis</h2>
 +
<ol>
 +
    <li>Cast an agarose gel and set it in the electrophoresis box.</li>
 +
    <li>Add 1 uL of 6x loading dye for every 5 uL of sample.</li>
 +
    <li>Load a DNA ladder into the first and last wells of the gel.</li>
 +
    <li>Load one sample into each remaining lane of the gel, being careful not to puncture the well or let the sample overflow.</li>
 +
    <li>Run gel at 120 V for approximately 25 minutes.</li>
 +
    <li>Image gel using the Ethidium Bromide protocol on the gel imager (we use Midori Green, but the EthBr protocol also works).</li>
 +
</ol>
 +
<h2>Plasmid Purification</h2>
 +
<ol>
 +
    <li>Grow up a colony overnight in 5 mL of LB in the 37 degree C shaker.</li>
 +
    <li>Follow the protocol included with the DNA mini-prep kit you are using. We used the Omega Bio-Tek E.Z.N.A. Plasmid DNA Mini Kit.</li>
 +
</ol>
 +
<h2>Double Digestion (dephos, dont automatically assume cutsmart, dpn1, consider glycerol)</h2>
 +
<ol>
 +
    <li>Use a Double Digest finder, such as that on the NEB website, to find the optimal buffer for your reaction.</li>
 +
    <li>Add the directed amount of your chosen buffer, and BSA if necessary</li>
 +
    <li>Add 0.75 uL of each enzyme.</li>
 +
    <li>Add DNA to be digested.</li>
 +
    <li>Bring up the total volume to 7.75 uL</li>
 +
</ol>
 
</html>
 
</html>

Revision as of 02:42, 19 September 2015

A. Using a Lab Notebook

  1. Always include the date and your initials or signature.
  2. Procedures from previous labs do not have to be rewritten, but should be clearly referenced.
  3. Always Include:
    • Any and all calculations
    • Number of cells seeded
    • Components of solutions
    • Type of cells
    • Type of media
    • Brand names of solutions used (Invitrogen, Bio-Rad, Promega, etc.)
    • Observations such as confluency, color of media, etc.
  4. Always write directly in the notebook, rather than writing it on a separate sheet and copying it later.
  5. Do no leave large open spaces. If you do, cross these out.
  6. If you cross out text, make sure it is still legible underneath.
  7. Number all pages in the notebook, and do not tear pages out.
  8. Write all entries in ink.
  9. Use original documentation when possible (e.g. machine printout rather than copying data by hand.)
  10. Be thorough! Somebody should be able to repeat your experiments by looking at the lab notebook.

Making LB

  1. Add 20 g of LB powder to 1 L of water and mix.
  2. Autoclave on a liquid cycle.
  3. If adding antibiotics, wait until the LB has cooled enough to comfortably touch the bottle. Otherwise, the heat will start to break them down.

Pouring plates

  1. Add 20 g of LB powder and 14 g of agar (NOT agarose) to 1 L of water and mix.
  2. Autoclave on a liquid cycle
  3. Wait until the LB agar has cooled enough to comfortably touch the bottle.
  4. If desired, add antibiotics now.
  5. Pour 10 - 20 mL of LB agar into each plate. This can be done by eye, but using a pipet-aid will give more consistent results
  6. If any bubbles formed on the surface of your plates, quickly pop them using a pipet tip.
  7. Allow the plates to solidify.
  8. Wrap plates in a bag, sleeve, or Parafilm.
  9. Store upside down at 4 degrees.

Transformation

  1. Take competent cells out of freezer and thaw on ice for 20-30 minutes. Take only as many as you need; they lose competency if re-frozen.
  2. Take agar plates out of storage and allow them to warm up to room temperature.
  3. Add 10 pg to 100 ng of DNA (usually about 1-5 uL depending on concentration) to 20 - 50 uL of competent cells.
  4. Mix tubes by gently flicking.
  5. Place tubes back on ice for 20-30 minutes.
  6. Heat shock transformation by placing it in 42 degree C water bath for 45 seconds.
    7. Note: Some strains of e coli may require shorter or longer heat shock times. Always check the strain before transforming!
  7. Set tubes back on ice for 2 minutes.
  8. Add 250 - 500 uL of LB and grow in the 37 shaking incubator for 45 minutes. If you are plating on ampicillin, this step can be skipped.
  9. Plate 50 uL of cells on each plate using beads or cell spreader.
  10. Grow overnight at 37 degrees C.

Making electrophoresis gels

  1. Add 1 g of agarose to 100 mL of 10x TAE buffer.
  2. Heat in the microwave for about 1 minute and 45 seconds, until the solution has come to a boil.
  3. Allow the liquid to cool for 5 minutes.
  4. Add 10 uL of Midori Green and swirl gently to mix.
  5. Pour 30 - 50 mL into gel tray with comb. Use as little gel as possible if you plan to extract and purify plasmids from it later.
  6. Let sit about 30 minutes to cast.
  7. Note: this makes a 1% gel, which works for most purposes. However, a lower percentage, such as 0.7% gel, is best for gel extractions, and as high as 2% agarose can be used when resolving large bands of DNA.

Gel Electrophoresis

  1. Cast an agarose gel and set it in the electrophoresis box.
  2. Add 1 uL of 6x loading dye for every 5 uL of sample.
  3. Load a DNA ladder into the first and last wells of the gel.
  4. Load one sample into each remaining lane of the gel, being careful not to puncture the well or let the sample overflow.
  5. Run gel at 120 V for approximately 25 minutes.
  6. Image gel using the Ethidium Bromide protocol on the gel imager (we use Midori Green, but the EthBr protocol also works).

Plasmid Purification

  1. Grow up a colony overnight in 5 mL of LB in the 37 degree C shaker.
  2. Follow the protocol included with the DNA mini-prep kit you are using. We used the Omega Bio-Tek E.Z.N.A. Plasmid DNA Mini Kit.

Double Digestion (dephos, dont automatically assume cutsmart, dpn1, consider glycerol)

  1. Use a Double Digest finder, such as that on the NEB website, to find the optimal buffer for your reaction.
  2. Add the directed amount of your chosen buffer, and BSA if necessary
  3. Add 0.75 uL of each enzyme.
  4. Add DNA to be digested.
  5. Bring up the total volume to 7.75 uL