Team:UIUC Illinois/Protocols

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   <div class="leftside">
   <div class="leftside">
     <ul>
     <ul>
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       <li><a onclick="showprot('#prot1')">Gel Eclectrophoresis</a></li>
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       <li><a onclick="showprot('#prot1')">Gel Electrophoresis</a></li>
       <li><a onclick="showprot('#prot2')">PCR Set-Up</a></li>
       <li><a onclick="showprot('#prot2')">PCR Set-Up</a></li>
       <li><a onclick="showprot('#prot3')">Bacterial Transformation</a></li>
       <li><a onclick="showprot('#prot3')">Bacterial Transformation</a></li>
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       <li><a onclick="showprot('#prot4')">Restriction Endonuclease Digest</a></li>
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       <li><a onclick="showprot('#prot4')">Endonuclease Digest</a></li>
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       <li><a onclick="showprot('#prot5')">CaCl2 Competent Cell Protocol</a></li>
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       <li><a onclick="showprot('#prot5')">CaCl2 Competence</a></li>
       <li><a onclick="showprot('#prot6')">Genomic DNA Purification</a></li>
       <li><a onclick="showprot('#prot6')">Genomic DNA Purification</a></li>
       <li><a onclick="showprot('#prot7')">Resting Cell Assay</a></li>
       <li><a onclick="showprot('#prot7')">Resting Cell Assay</a></li>
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   <div class="rightside">
   <div class="rightside">
     <div id="prot1" class="protocolactive">
     <div id="prot1" class="protocolactive">
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       <a href="http://www.addgene.org/plasmid-protocols/gel-electrophoresis/"><h2> Gel Electrophoresis</h2></a>
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       <a href="http://www.addgene.org/plasmid-protocols/gel-electrophoresis/"><h2> Gel Electrophoresis</h2></a>  
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      <p>Summarize Protocol Here</p>   
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     </div>
     </div>
     <div id="prot2" class="protocol">
     <div id="prot2" class="protocol">
       <a href="https://www.neb.com/protocols/1/01/01/pcr-protocol-m0530"><h2> PCR Set-Up</h2></a>
       <a href="https://www.neb.com/protocols/1/01/01/pcr-protocol-m0530"><h2> PCR Set-Up</h2></a>
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      <p>Summary Paragraph</p>
 
</div>
</div>
<div id="prot3" class="protocol">
<div id="prot3" class="protocol">
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       <a href="http://www.addgene.org/plasmid-protocols/bacterial-transformation/"><h2> Bacterial Transformation</h2></a>
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       <a href="http://www.addgene.org/plasmid-protocols/bacterial-transformation/"><h2> Bacterial Transformation</h2></a>  
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      <p>Summarize Protocol Here</p>   
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     </div>
     </div>
<div id="prot4" class="protocol">
<div id="prot4" class="protocol">
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       <a href="https://www.neb.com/protocols/2012/12/07/optimizing-restriction-endonuclease-reactions"><h2> Restriction Endonuclease Digest</h2></a>
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       <a href="https://www.neb.com/protocols/2012/12/07/optimizing-restriction-endonuclease-reactions"><h2> Endonuclease Digest</h2></a>
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      <p>Summary Paragraph</p>
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</div>
</div>
<div id="prot5" class="protocol">
<div id="prot5" class="protocol">
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       <a href="http://www.med.nyu.edu/medicine/labs/blaserlab/Protocols/Ecoli_competent_cells_protocol_&_transformation.pdf"><h2> CaCl2 Competent Cell Protocol</h2></a>
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       <a href="http://www.flemingtonlab.com/Protocols/CompCellsCaCl2Method.pdf"><h2> CaCl2 Competence</h2></a>  
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      <p>Summarize Protocol Here</p>   
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     </div>
     </div>
<div id="prot6" class="protocol">
<div id="prot6" class="protocol">
       <a href="http://www.promega.com/~/media/files/resources/protcards/wizard%20genomic%20dna%20purification%20kit%20quick%20protocol.pdf"><h2> Genomic DNA Purification</h2></a>
       <a href="http://www.promega.com/~/media/files/resources/protcards/wizard%20genomic%20dna%20purification%20kit%20quick%20protocol.pdf"><h2> Genomic DNA Purification</h2></a>
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      <p>Summary Paragraph</p>
 
</div>
</div>
<div id="prot7" class="protocol">
<div id="prot7" class="protocol">
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       <a href="http://pubs.acs.org/doi/pdf/10.1021/sb4000146"><h2> Resting Cell Assay</h2></a>
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       <a href="http://pubs.acs.org/doi/pdf/10.1021/sb4000146"><h2> Resting Cell Assay</h2></a>  
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      <p>Summarize Protocol Here</p>   
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     </div>
     </div>
<div id="prot8" class="protocol">
<div id="prot8" class="protocol">
       <a href="http://www.csun.edu/~mls42367/Protocols/Colony%20PCR.pdf"><h2> Colony PCR</h2></a>
       <a href="http://www.csun.edu/~mls42367/Protocols/Colony%20PCR.pdf"><h2> Colony PCR</h2></a>
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      <p>Summary Paragraph</p>
 
</div>
</div>
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<ol>
<ol>
<li>Inoculate 100mL MRS with 1mL of an overnight culture and incubate at 37C until it reaches an OD600 = 0.5 – 0.8.<br>
<li>Inoculate 100mL MRS with 1mL of an overnight culture and incubate at 37C until it reaches an OD600 = 0.5 – 0.8.<br>
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<p style="text-indent:30px;">
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<p style="text-indent:30px;">a. Pre-warm the MRS for faster growth</p><br>
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a. Pre-warm the MRS for faster growth
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<p style="text-indent:30px;">b. Take 3.5-5.5 hours to get to 0.5</p><br>
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b. Take 3.5-5.5 hours to get to 0.5
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<p style="text-indent:30px;">c. All liquid cultures can be done outside the anaerobic chamber; however, do not use a shaker</p><br>
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c. All liquid cultures can be done outside the anaerobic chamber; however, do not use a shaker
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</li>
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</p>
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 +
<li>Centrifuge culture at 4000rpm for 10min at 4C and wash with cold 3.5X SMEB.  Centrifuge in 2, 50 ml conical tubes<br>
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<p style="text-indent:30px;">a. Resuspend each pellet in 20 ml 3.5X SMEB (40 ml total)</p><br>
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<p style="text-indent:30px;">b. Can combine into one tube if desired but use 40 ml per wash</p><br>
</li>
</li>
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<li>Centrifuge culture at 4000rpm for 10min at 4C and wash with cold 3.5X SMEB.  Centrifuge in 2, 50 ml conical tubes<br>
 
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<p style="text-indent:30px;">
 
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a. Resuspend each pellet in 20 ml 3.5X SMEB (40 ml total)
 
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b. Can combine into one tube if desired but use 40 ml per wash
 
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</p></li>
 
<li>Repeat step 2 at least twice for three total washes.</li><br>
<li>Repeat step 2 at least twice for three total washes.</li><br>
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<li>Resuspend cells in 1.0mL 3.5X SMEB (concentrate 100 fold).<br>
<li>Resuspend cells in 1.0mL 3.5X SMEB (concentrate 100 fold).<br>
<p style="text-indent:30px;">a. Mix well using a pipettor</p>
<p style="text-indent:30px;">a. Mix well using a pipettor</p>
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</li>
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</li><br>
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<li>Transfer 0.2mL cells to a microfuge tube and add DNA; mix and transfer to a cold 0.2cm cuvette.<br></li>
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<li>Transfer 0.2mL cells to a microfuge tube and add DNA; mix and transfer to a cold 0.2cm cuvette.<br></li><br>
<li>Electroporate at 2.45kV, 25μFD, 200Ω.<br>
<li>Electroporate at 2.45kV, 25μFD, 200Ω.<br>
<p style="text-indent:30px;">a. We now use an eppendorf electoporator that has only a voltage setting (we use 2.5 kV; typical time constant is ~3.3)</p>
<p style="text-indent:30px;">a. We now use an eppendorf electoporator that has only a voltage setting (we use 2.5 kV; typical time constant is ~3.3)</p>
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</li>
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</li><br>
<li>Transfer cells (gently) to 3.0mL MRS and incubate at 37C.<br>
<li>Transfer cells (gently) to 3.0mL MRS and incubate at 37C.<br>
-
<p style="text-indent:30px;">a. ~16 hours is sufficient
+
<p style="text-indent:30px;">a. ~16 hours is sufficient</p><br>
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b. Does not need to be in anaerobic shaker but no shaking
+
<p style="text-indent:30px;">b. Does not need to be in anaerobic shaker but no shaking</p><br>
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c. We use small culture tubes but you could use falcon tubes
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<p style="text-indent:30px;">c. We use small culture tubes but you could use falcon tubes</p><br>
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</p></li>
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</li>
<li>Dilute cells accordingly and plate on MRS plus appropriate antibiotic.<br>
<li>Dilute cells accordingly and plate on MRS plus appropriate antibiotic.<br>
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<p style="text-indent:30px;">a. L. gasseri ATCC 33323 – 5.0 g/ml erythromycin
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<p style="text-indent:30px;">a. L. gasseri ATCC 33323 – 5.0 ug/ml erythromycin</p><br>
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b. Make sure you plate a negative control
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<p style="text-indent:30px;">b. Make sure you plate a negative control</p><br>
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c. I usually use add 40l and 200l on two plates each
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<p style="text-indent:30px;">c. I usually use add 40ml and 200ml on two plates each</p><br>
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</p></li>
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</li>
<li>Incubate 2-3 days in anaerobic chamber at 37C<br></li>
<li>Incubate 2-3 days in anaerobic chamber at 37C<br></li>
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Latest revision as of 03:51, 18 October 2014


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Lactobacillus Transformation

  1. Inoculate 100mL MRS with 1mL of an overnight culture and incubate at 37C until it reaches an OD600 = 0.5 – 0.8.

    a. Pre-warm the MRS for faster growth


    b. Take 3.5-5.5 hours to get to 0.5


    c. All liquid cultures can be done outside the anaerobic chamber; however, do not use a shaker


  2. Centrifuge culture at 4000rpm for 10min at 4C and wash with cold 3.5X SMEB. Centrifuge in 2, 50 ml conical tubes

    a. Resuspend each pellet in 20 ml 3.5X SMEB (40 ml total)


    b. Can combine into one tube if desired but use 40 ml per wash


  3. Repeat step 2 at least twice for three total washes.

  4. Resuspend cells in 1.0mL 3.5X SMEB (concentrate 100 fold).

    a. Mix well using a pipettor


  5. Transfer 0.2mL cells to a microfuge tube and add DNA; mix and transfer to a cold 0.2cm cuvette.

  6. Electroporate at 2.45kV, 25μFD, 200Ω.

    a. We now use an eppendorf electoporator that has only a voltage setting (we use 2.5 kV; typical time constant is ~3.3)


  7. Transfer cells (gently) to 3.0mL MRS and incubate at 37C.

    a. ~16 hours is sufficient


    b. Does not need to be in anaerobic shaker but no shaking


    c. We use small culture tubes but you could use falcon tubes


  8. Dilute cells accordingly and plate on MRS plus appropriate antibiotic.

    a. L. gasseri ATCC 33323 – 5.0 ug/ml erythromycin


    b. Make sure you plate a negative control


    c. I usually use add 40ml and 200ml on two plates each


  9. Incubate 2-3 days in anaerobic chamber at 37C

The Institute for Genomic Biology
Biosystems Design, South wing, 2nd floor
University of Illinois at Urbana-Champaign
1206 West Gregory Drive
61801 Urbana, Illinois
illinoisigem@gmail.com
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