Difference between revisions of "Team:McMasterU/CDifficile"

(Created page with "<h1>Compiled Protocols for TcdC Plasmids Design</h1> <br><br> <h2>I. C. Difficile gDNA Extraction Using Phenolchloroform</h2> <br> <h3>Materials</h3> <br> <strong>Resuspensi...")
 
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<li>DNA samples were diluted to 125 ng/uL</li>
 
<li>DNA samples were diluted to 125 ng/uL</li>
 
<li>The following master mix solution was prepared</li>
 
<li>The following master mix solution was prepared</li>
 +
 +
<table style="width:100%>
 +
<tr>
 +
  <th>Reagent</th>
 +
  <th>1x(uL)</th>
 +
</tr>
 +
<tr>
 +
  <td>Forward Primer</td>
 +
  <td>2.5</td>
 +
</tr>
 +
<tr>
 +
  <td>Reverse Primer</td>
 +
  <td>2.5</td>
 +
</tr>
 +
<tr>
 +
  <td>dNTP</td>
 +
  <td>1</td>
 +
</tr>
 +
<tr>
 +
  <td>Phusion Buffer</td>
 +
  <td>10</td>
 +
</tr>
 +
<tr>
 +
  <td>Phusion High Fidelity Polymerase</td>
 +
  <td>0.5</td>
 +
</tr>
 +
<tr>
 +
  <td>Nuclease-free water</td>
 +
  <td>31.5</td>
 +
</tr>
 +
</table>
 +
<br>
 +
 +
<li>2 uL of DNA was added to 48 uL of the master mix</li>
 +
<li>PCR reaction was run in a thermocycler using the following conditions for 35 cycles:</li>
 +
 +
<table>
 +
<tr>
 +
  <th>Step</th>
 +
  <th>Temperature (C)</th>
 +
  <th>Time</th>
 +
</tr>
 +
<tr>
 +
  <td>Initial Denaturing</td>
 +
  <td>98</td>
 +
  <td>10 minutes</td>
 +
</tr>
 +
<tr>
 +
  <td>Denaturing</td>
 +
  <td>98</td>
 +
  <td>30 seconds</td>
 +
</tr>
 +
<tr>
 +
  <td>Annealing</td>
 +
  <td>50</td>
 +
  <td>20 seconds</td>
 +
</tr>
 +
<tr>
 +
  <td>Elongation</td>
 +
  <td>72</td>
 +
  <td>20 seconds</td>
 +
</tr>
 +
<tr>
 +
  <td>Final elongation</td>
 +
  <td>72</td>
 +
  <td>10 minutes</td>
 +
</tr>
 +
<tr>
 +
  <td>Hold</td>
 +
  <td>4</td>
 +
  <td>Indefinite</td>
 +
</tr>
 +
</table>
 +
 +
<li>The PCR reactions were run on a 1% agarose gel and purified using gel extraction <i>(see Other Protocols)</i></li>
 +
</ol>
 +
 +
<br><br>
 +
<h2>Digestion of TcdC and pET15b</h2>
 +
<br>
 +
<h3>Protocol</h3>
 +
 +
<ol>
 +
<li>The following reaction was set up:</li>
 +
<table>
 +
<tr>
 +
  <th>Component</th>
 +
  <th>50 uL Reaction </th>
 +
</tr>
 +
<tr>
 +
  <td>DNA</td>
 +
  <td>1 ug</td>
 +
</tr>
 +
<tr>
 +
  <td>FastDigest Buffer</td>
 +
  <td>5 uL</td>
 +
</tr>
 +
<tr>
 +
  <td>Ndel</td>
 +
  <td>1.0 uL</td>
 +
</tr>
 +
<tr>
 +
  <td>BamHI</td>
 +
  <td>1.0 uL</td>
 +
</tr>
 +
<tr>
 +
  <td>Nuclease free water</td>
 +
  <td>To 50 uL</td>
 +
</tr>
 +
</table>
 +
 +
<li>Reactions were incubated at 37 degrees Celsius for one hour</li>
 +
<li>Digestion reaction were run on a 1% agarose gel and purified using gel extraction <i>(see Other Protocols)</i></li>
 +
</ol>
 +
<br><br>
 +
 +
<h2>Ligation of TcdC to pET15b</h2>
 +
<h3>Protocol</h3>
 +
<ol>
 +
<li>The following reaction was set up:</li>
 +
 +
<table>
 +
<tr>
 +
  <th>Component</th>
 +
  <th>20 uL Reaction</th>
 +
</tr>
 +
<tr>
 +
  <td>10X T4 Ligase Buffer</td>
 +
  <td>2 uL</td>
 +
</tr>
 +
<tr>
 +
  <td>Vector DNA</td>
 +
  <td>70.54 ng (0.02 pmol)</td>
 +
</tr>
 +
<tr>
 +
  <td>Insert DNA</td>
 +
  <td>25.81 ng (0.06 pmol)</td>
 +
</tr>
 +
<tr>
 +
  <td>T4 DNA Ligase</td>
 +
  <td>1 uL</td>
 +
</tr>
 +
<tr>
 +
  <td>Nuclease-free water</td>
 +
  <td>To 20 uL</td>
 +
</tr>
 +
</table>
 +
 +
<li>The ligation reactions were incubated at 16 degrees Celsius overnight.</li>
 +
</ol>
 +
<br><br>
 +
 +
<h2>V. Transformation Using Chemically Competent Cells</h2>
 +
<h3>Protocol</h3>
 +
 +
<ol>
 +
<li>Invitrogen DH5 alpha cells were thawed on ice for 10 minutes, 50 uL of cells were pipetted into transformation tube</li>
 +
<li>5 uL of ligation reaction was added into cell mixture</li>
 +
<li>Mixture was incubated on ice for 30 minutes</li>
 +
<li>Mixture was heat shocked at 42 C for 30 seconds</li>
 +
<li>Mixture was transferred to ice for 5 minutes.</li>
 +
<li>950 uL of LB broth was added to mixture</li>
 +
<li>Mixture was incubated at 37 C for 60 minutes, shaking at 250 rpm</li>
 +
<li>Selection plates containing 50 ug/mL of ampicillin were warmed to 37 C</li>
 +
<li>Cells were pelleted at 4000 g for 1 minute</li>
 +
<li>900 uL of LB broth was removed </li>
 +
<li>Cells were plated onto selection plates and incubated for 16 hours at 37 C</li>
 +
<li>Colonies were picked and grown in 5 mL of LB broth for 16-18 hours </li>
 +
</ol>
 +
<br><br>
 +
 +
<h2>VI. Miniprep Plasmids</h2>
 +
<p>Promega Miniprep Kit was used.</p>
 +
<ol>
 +
<li>1.5 mL of liquid culture was centrifuged at maximum speed for 30 seconds. Supernatant was discarded</li>
 +
<li>An additional 1.5 mL of bacterial culture was added to the same tube and step 1 was repeated.</li>
 +
<li>600 uL of nuclease-free water was used to resuspend the cells</li>
 +
<li>100 uL of cell lysis buffer was added and mixed by inverting the tube 6 times</li>
 +
<li>350 uL of cold neutralization solution was added</li>
 +
<li>Mixture was centrifuged at maximum speed for 3 minutes</li>
 +
<li>Supernatant (~900 uL) was transferred to a minicolumn without disturbing the cell debris pellet and centrifuged at maximum</li>
 +
<li>speed for 15 seconds. Supernatant was discarded.</li>
 +
<li>200 uL of endotoxin removal wash was added to the minicolumn. Minicolumn was centrifuged at maximum speed for 15 seconds</li>
 +
<li>400 uL of column wash solution was added to the minicolumn and centrifuged for 30 seconds</li>
 +
<li>Minicolumn was transferred to a clean 1.5 mL Eppendorf tube and 30 uL of elution buffer was added</li>
 +
<li>After 1 minute, tubes were centrifuged for 15 seconds to elute the plasmid DNA. </li>
 +
</ol>
 +
 +
<h3>Other Protocols</h3>
 +
<i>Ethanol Precipitation</i>
 +
<ol>
 +
<li>Add 0.1 volumes of 3M NaOAc, 2.5 volumes of ice cold 100% ethanol, and mix</li>
 +
<li>Precipitate at -20 C for at least one hour </li>
 +
<li>Centrifuge at 15000 rpm at 4 C for 30 minutes</li>
 +
<li>Wash pellet twice with 0.5 mL of ice cold 75% ethanol, spinning at 4 C for 10 minutes each time</li>
 +
<li>Remove ethanol and dry the pellet in a Speed-Vac</li>
 +
<li>Resuspend pellet in nuclease free water</li>
 +
</ol>
 +
<br>
 +
 +
<i>Agarose Gel Electrophoresis</i>
 +
<ol>
 +
<li>Dilute 50X TAE to 1X</li>
 +
<li>Add 1% mass of agarose to TAE</li>
 +
<li>Microwave until agarose has dissolved</li>
 +
<li>Cool to around 50 C, then add SYBR Safe (1 uL for every 10 mL of TAE)</li>
 +
<li>Pour gel into casting tray with a comb</li>
 +
<li>Once gel has solidified, place the gel into a gel electrophoresis machine and add 1X TAE until the gel is fully covered</li>
 +
<li>Load 5 uL of 1kb DNA ladder into the first well and DNA samples into subsequent wells</li>
 +
<li>Run the gel for about 45 minutes</li>
 +
</ol>
 +
<br>
 +
 +
<i>DNA Extraction from Agarose Gels</i>
 +
<p>Nucleospin Gel and PCR Clean-up Kit was used</p>
 +
<ol>
 +
<li>Use a clean razor blade to excise DNA fragment from gel</li>
 +
<li>For every 100 mg of gel, add 200 uL of Buffer NTI (gel solubilization solution)</li>
 +
<li>Place a binding column into a collection tube and add 700 uL of sample. Centrifuge for 30 seconds at 11,000 g. Discard flow through. Repeat if necessary</li>
 +
<li>Add 700 uL of Buffer NT3 (wash buffer). Centrifuge for 30 seconds at 11,000 g. Discard flow-through. Repeat this step to minimize chaotropic salt carry-over.</li>
 +
<li>Centrifuge for 1 minute at 11,000 g to remove Buffer NT3 completely. To remove residual ethanol, incubate the columns for 5 minutes at 70 C prior to elution</li>
 +
<li>Place the column into a new 1.5 mL Eppendorf tube and add 30 uL of nuclease-free water. Incubate for 1 minute before centrifuging for 1 minute at 11,000 g.</li>
 +
</ol>

Revision as of 09:11, 27 October 2017

Compiled Protocols for TcdC Plasmids Design



I. C. Difficile gDNA Extraction Using Phenolchloroform


Materials


Resuspension Buffer

    10 mM Tris-HCl, pH 8.0
    20% (w/v) Sucrose

Lysis Buffer

    10 mM Tris-HCl, pH 8.0
    1mM EDTA
    1% (w/v) SDS

RNase A (10mg/mL)

    10 mg bovine pancreating RNase A
    10 mM Tris, pH 8.0
    500 mM NaCl
    850 uL water
    Heated to 95 C until use



Protocol

  1. 10 mL of overnight culture was pelleted at 5000 rpm for 5 minutes
  2. Pellets were resuspended in 1 mL of resuspension buffer.
  3. 9 mL of lysis buffer was added to resuspended pellet.
  4. Cell suspension was incubated at 37 C for 1 hour, shaking at 260 rpm.
  5. Crude cell lysate was extracted with an equal volume of phenol chloroform.
  6. Aqueous phase was recovered to a new tube and 25 ug RNase A was added, crude lysate was incubated at 37 C for 20 minutes.
  7. Crude lysate was extracted with equal volume of phenolchloroform, aqueous phase removed to a new tube.
  8. DNA was precipitated using ethanol precipitation. (see Other Protocols)
  9. DNA was dissolved in 1.5 mL nuclease free water



II. PCR Amplification of BAA-1870, BAA-1875, and WT TcdC


Protocol

  1. Primers were diluted to 10 uM
  2. dNTP was diluted to 10 mM
  3. DNA samples were diluted to 125 ng/uL
  4. The following master mix solution was prepared
    5. Reagent 1x(uL)
    Forward Primer 2.5
    Reverse Primer 2.5
    dNTP 1
    Phusion Buffer 10
    Phusion High Fidelity Polymerase 0.5
    Nuclease-free water 31.5


  5. 2 uL of DNA was added to 48 uL of the master mix
  6. PCR reaction was run in a thermocycler using the following conditions for 35 cycles:
    7. Step Temperature (C) Time
    Initial Denaturing 98 10 minutes
    Denaturing 98 30 seconds
    Annealing 50 20 seconds
    Elongation 72 20 seconds
    Final elongation 72 10 minutes
    Hold 4 Indefinite
  7. The PCR reactions were run on a 1% agarose gel and purified using gel extraction (see Other Protocols)



Digestion of TcdC and pET15b


Protocol

  1. The following reaction was set up:
    2. Component 50 uL Reaction
    DNA 1 ug
    FastDigest Buffer 5 uL
    Ndel 1.0 uL
    BamHI 1.0 uL
    Nuclease free water To 50 uL
  2. Reactions were incubated at 37 degrees Celsius for one hour
  3. Digestion reaction were run on a 1% agarose gel and purified using gel extraction (see Other Protocols)



Ligation of TcdC to pET15b

Protocol

  1. The following reaction was set up:
    2. Component 20 uL Reaction
    10X T4 Ligase Buffer 2 uL
    Vector DNA 70.54 ng (0.02 pmol)
    Insert DNA 25.81 ng (0.06 pmol)
    T4 DNA Ligase 1 uL
    Nuclease-free water To 20 uL
  2. The ligation reactions were incubated at 16 degrees Celsius overnight.



V. Transformation Using Chemically Competent Cells

Protocol

  1. Invitrogen DH5 alpha cells were thawed on ice for 10 minutes, 50 uL of cells were pipetted into transformation tube
  2. 5 uL of ligation reaction was added into cell mixture
  3. Mixture was incubated on ice for 30 minutes
  4. Mixture was heat shocked at 42 C for 30 seconds
  5. Mixture was transferred to ice for 5 minutes.
  6. 950 uL of LB broth was added to mixture
  7. Mixture was incubated at 37 C for 60 minutes, shaking at 250 rpm
  8. Selection plates containing 50 ug/mL of ampicillin were warmed to 37 C
  9. Cells were pelleted at 4000 g for 1 minute
  10. 900 uL of LB broth was removed
  11. Cells were plated onto selection plates and incubated for 16 hours at 37 C
  12. Colonies were picked and grown in 5 mL of LB broth for 16-18 hours



VI. Miniprep Plasmids

Promega Miniprep Kit was used.

  1. 1.5 mL of liquid culture was centrifuged at maximum speed for 30 seconds. Supernatant was discarded
  2. An additional 1.5 mL of bacterial culture was added to the same tube and step 1 was repeated.
  3. 600 uL of nuclease-free water was used to resuspend the cells
  4. 100 uL of cell lysis buffer was added and mixed by inverting the tube 6 times
  5. 350 uL of cold neutralization solution was added
  6. Mixture was centrifuged at maximum speed for 3 minutes
  7. Supernatant (~900 uL) was transferred to a minicolumn without disturbing the cell debris pellet and centrifuged at maximum
  8. speed for 15 seconds. Supernatant was discarded.
  9. 200 uL of endotoxin removal wash was added to the minicolumn. Minicolumn was centrifuged at maximum speed for 15 seconds
  10. 400 uL of column wash solution was added to the minicolumn and centrifuged for 30 seconds
  11. Minicolumn was transferred to a clean 1.5 mL Eppendorf tube and 30 uL of elution buffer was added
  12. After 1 minute, tubes were centrifuged for 15 seconds to elute the plasmid DNA.

Other Protocols

Ethanol Precipitation

  1. Add 0.1 volumes of 3M NaOAc, 2.5 volumes of ice cold 100% ethanol, and mix
  2. Precipitate at -20 C for at least one hour
  3. Centrifuge at 15000 rpm at 4 C for 30 minutes
  4. Wash pellet twice with 0.5 mL of ice cold 75% ethanol, spinning at 4 C for 10 minutes each time
  5. Remove ethanol and dry the pellet in a Speed-Vac
  6. Resuspend pellet in nuclease free water


Agarose Gel Electrophoresis

  1. Dilute 50X TAE to 1X
  2. Add 1% mass of agarose to TAE
  3. Microwave until agarose has dissolved
  4. Cool to around 50 C, then add SYBR Safe (1 uL for every 10 mL of TAE)
  5. Pour gel into casting tray with a comb
  6. Once gel has solidified, place the gel into a gel electrophoresis machine and add 1X TAE until the gel is fully covered
  7. Load 5 uL of 1kb DNA ladder into the first well and DNA samples into subsequent wells
  8. Run the gel for about 45 minutes


DNA Extraction from Agarose Gels

Nucleospin Gel and PCR Clean-up Kit was used

  1. Use a clean razor blade to excise DNA fragment from gel
  2. For every 100 mg of gel, add 200 uL of Buffer NTI (gel solubilization solution)
  3. Place a binding column into a collection tube and add 700 uL of sample. Centrifuge for 30 seconds at 11,000 g. Discard flow through. Repeat if necessary
  4. Add 700 uL of Buffer NT3 (wash buffer). Centrifuge for 30 seconds at 11,000 g. Discard flow-through. Repeat this step to minimize chaotropic salt carry-over.
  5. Centrifuge for 1 minute at 11,000 g to remove Buffer NT3 completely. To remove residual ethanol, incubate the columns for 5 minutes at 70 C prior to elution
  6. Place the column into a new 1.5 mL Eppendorf tube and add 30 uL of nuclease-free water. Incubate for 1 minute before centrifuging for 1 minute at 11,000 g.