Difference between revisions of "Team:Aachen/Lab/Protocols"

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        <div class="single_header_title">
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<div class="single_header_title">
            <h1>Protocols</h1>
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<h1>Protocols</h1>
        </div>
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</div>
      <h2 style="border-bottom: 5px solid #005b04;padding-left: 0.3cm;">General</h2>
+
 
    <br/>
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<h2 style="border-bottom: 5px solid #005b04;padding-left: 0.3cm;">General</h2>
        <ol id="category-tabs" style=" padding-left:0.1cm; list-style-type: none;">
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<br/>
            <li><a href="#" style="text-decoration: none; font-size: 18px">
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<ol id="category-tabs" style=" padding-left:0.1cm; list-style-type: none;">
              <b>Polymerase Chain Reaction (PCR)</b>&nbsp; <i class="fa fa-chevron-circle-down"></i></a>
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<li><a href="#" style="text-decoration: none; font-size: 18px">
                <ol  class="protocolhide" style=" padding-left: 0.1cm; list-style-type: none;">
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<b>Polymerase Chain Reaction (PCR)</b>&nbsp; <i class="fa fa-chevron-circle-down"></i></a>
                    <li >Colony-PCR using Alkaline PEG</li>
+
<ol  class="protocolhide" style=" padding-left: 0.1cm; list-style-type: none;">
                    <li> Colony-PCR with Heatshock</li>
+
<li >Colony-PCR using Alkaline PEG</li>
                    <li>Extended Colony-PCR</li>
+
<li> Colony-PCR with Heatshock</li>
                </ol>
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<li>Extended Colony-PCR</li>
            </li>
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</ol>
  <li><a href="#" style="text-decoration: none; font-size: 18px">
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</li>
 +
<li><a href="#" style="text-decoration: none; font-size: 18px">
 
<b>Precipitation</b>&nbsp;<i class="fa fa-chevron-circle-down"></i></a>
 
<b>Precipitation</b>&nbsp;<i class="fa fa-chevron-circle-down"></i></a>
 
<ol  class="protocolhide" style=" padding-left: 0.1cm; list-style-type: none;">
 
<ol  class="protocolhide" style=" padding-left: 0.1cm; list-style-type: none;">
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<li  style=" padding-right: 0.3cm;"><span>conditions:</span></li>
 
<li  style=" padding-right: 0.3cm;"><span>conditions:</span></li>
 
<li style=" padding-right: 0.3cm;"><span>time:&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;3 min</span></li>
 
<li style=" padding-right: 0.3cm;"><span>time:&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;3 min</span></li>
<li style=" padding-right: 0.3cm;"><span>pulse on: &nbsp;&nbsp;&nbsp;&nbsp;30 s</span></li>
+
<li style=" padding-right: 0.3cm;"><span>pulse on:&nbsp;&nbsp;&nbsp;&nbsp;30 s</span></li>
<li style=" padding-right: 0.3cm;"><span>pulse off: &nbsp;&nbsp;&nbsp;&nbsp;20 s</span></li>
+
<li style=" padding-right: 0.3cm;"><span>pulse off:&nbsp;&nbsp;&nbsp;&nbsp;20 s</span></li>
 
<li style=" padding-right: 0.3cm;"><span>amplitude:&nbsp;&nbsp;50%</span></li>
 
<li style=" padding-right: 0.3cm;"><span>amplitude:&nbsp;&nbsp;50%</span></li>
  
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 +
<li><a href="#" style="text-decoration: none; font-size: 18px">
 +
<b>Transformation</b>&nbsp;<i class="fa fa-chevron-circle-down"></i></a>
 +
<ol  class="protocolhide" style=" padding-left:0.1cm; padding-right:0.1cm;list-style-type: none;">
 +
<li ><b>E. coli</b>
 +
<ol type="1" style=" style=" padding-left:0.1cm; padding-right:0.1cm;align-content: center;">
 +
<li  style=" padding-right: 0.3cm;"><span>Thaw 100 µL <b>E. coli DH5α</b> or <b>E. coli BL21(DE3)</b> Gold on ice</span></li>
 +
<li style=" padding-right: 0.3cm;"><span>Add <b>DNA</b> (2-400 ng/µL: 50 ng if Plasmid DNA; 2 µL (~20 ng) ligation mixture;
 +
1-4 µL PLICing-reaction) to the competent cells and swirl gently</span></li>
 +
<li style=" padding-right: 0.3cm;"><span>Incubate for <b>15-30 min on ice.</b></span></li>
 +
<li style=" padding-right: 0.3cm;"><span>Perform heatshock of the competent cells using a preheated water bath at <b>42°C for 45s</b> (DH5α, BL21).</li>
 +
<li style=" padding-right: 0.3cm;"><span>Samples were immediately cooled down <b>5 min on ice.</b></span></li>
 +
<li style=" padding-right: 0.3cm;"><span>Fill up the transformation mix <b>to 1 mL with SOC media</b> and
 +
incubate at <b>37°C at 250rpm for 45 min </b>(45-60 min) for recovery of the cells.</span></li>
 +
<li style=" padding-right: 0.3cm;"><span><b>Plate cells</b> on LB agar plate with antibiotic respectively and dry them under the clean bench.
 +
<ol type="i" style=" padding-left: 0.5cm">
 +
<li><span>200 µL</span></li>
 +
<li><span>resuspended pellet (from centrifugation of the leftover)</span></li>
 +
</ol>
 +
</span></li>
 +
<li style=" padding-right: 0.3cm;"><span>Incubate the agar plates at <b>37°C overnight</b>(16-18 hours).</span></li>
 +
<li><span>Count single colonies of each agar plate on the next day.</span></li>
  
 +
</ol></li>
 +
<li><b>Sonication</b>
  
 +
<ol type="1"style=" style=" padding-left:0.1cm; padding-right:0.1cm; align-content: center" >
 +
<li style=" padding-right: 0.3cm;"><span>Lorem ipsum dolor sit amet, consectetuer adipiscing elit.</span></li>
 +
<li style=" padding-right: 0.3cm;"><span>Maecenas porttitor congue massa.</span></li>
 +
<li style=" padding-right: 0.3cm;"><span>Fusce posuere, magna sed pulvinar ultricies,
 +
purus lectus malesuada libero, sit amet commodo magna eros quis urna.</b></span></li>
 +
<li style=" padding-right: 0.3cm;"><span>Nunc viverra imperdiet enim. Fusce est.</span></li>
 +
<li style=" padding-right: 0.3cm;"><span>Nunc viverra imperdiet enim. Fusce est.</span></li>
 +
<li style=" padding-right: 0.3cm;"><span>Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas.
 +
<ol type="i" style=" padding-left: 0.5cm">
 +
<li><span>Proin pharetra nonummy pede.</span></li>
 +
<li><span>Mauris et orci.</span></li>
 +
</ol>
 +
</span></li>
  
            <li><a href="#" style="text-decoration: none; font-size: 18px">
 
                <b>Transformation</b>&nbsp;<i class="fa fa-chevron-circle-down"></i></a>
 
                <ol  class="protocolhide" style=" padding-left:0.1cm; padding-right:0.1cm;list-style-type: none;">
 
                    <li ><b>E. coli</b>
 
                        <ol type="1" style=" style=" padding-left:0.1cm; padding-right:0.1cm;align-content: center;">
 
                        <li  style=" padding-right: 0.3cm;"><span>Thaw 100 µL <b>E. coli DH5α</b> or <b>E. coli BL21(DE3)</b> Gold on ice</span></li>
 
                        <li style=" padding-right: 0.3cm;"><span>Add <b>DNA</b> (2-400 ng/µL: 50 ng if Plasmid DNA; 2 µL (~20 ng) ligation mixture;
 
                        1-4 µL PLICing-reaction) to the competent cells and swirl gently</span></li>
 
                      <li style=" padding-right: 0.3cm;"><span>Incubate for <b>15-30 min on ice.</b></span></li>
 
                      <li style=" padding-right: 0.3cm;"><span>Perform heatshock of the competent cells using a preheated water bath at <b>42°C for 45s</b> (DH5α, BL21).</li>
 
                      <li style=" padding-right: 0.3cm;"><span>Samples were immediately cooled down <b>5 min on ice.</b></span></li>
 
                      <li style=" padding-right: 0.3cm;"><span>Fill up the transformation mix <b>to 1 mL with SOC media</b> and
 
                        incubate at <b>37°C at 250rpm for 45 min </b>(45-60 min) for recovery of the cells.</span></li>
 
                      <li style=" padding-right: 0.3cm;"><span><b>Plate cells</b> on LB agar plate with antibiotic respectively and dry them under the clean bench.
 
                          <ol type="i" style=" padding-left: 0.5cm">
 
                              <li><span>200 µL</span></li>
 
                              <li><span>resuspended pellet (from centrifugation of the leftover)</span></li>
 
                          </ol>
 
                      </span></li>
 
                    <li style=" padding-right: 0.3cm;"><span>Incubate the agar plates at <b>37°C overnight</b>(16-18 hours).</span></li>
 
                    <li><span>Count single colonies of each agar plate on the next day.</span></li>
 
  
                    </ol></li>
+
</ol></li>
                    <li><b>Sonication</b>
+
  
                        <ol type="1"style=" style=" padding-left:0.1cm; padding-right:0.1cm; align-content: center" >
+
<li>Saccharomyces </li>
                            <li style=" padding-right: 0.3cm;"><span>Lorem ipsum dolor sit amet, consectetuer adipiscing elit.</span></li>
+
</ol>
                            <li style=" padding-right: 0.3cm;"><span>Maecenas porttitor congue massa.</span></li>
+
</li>
                            <li style=" padding-right: 0.3cm;"><span>Fusce posuere, magna sed pulvinar ultricies,
+
<li><a href="#" style="text-decoration: none; font-size: 18px">
                                purus lectus malesuada libero, sit amet commodo magna eros quis urna.</b></span></li>
+
<b>Preparation of Chemical Competent Cells </b>&nbsp;<i class="fa fa-chevron-circle-down"></i></a>
                            <li style=" padding-right: 0.3cm;"><span>Nunc viverra imperdiet enim. Fusce est.</span></li>
+
<ol  class="protocolhide" style=" padding-left: 0.1cm; list-style-type: none;">
                            <li style=" padding-right: 0.3cm;"><span>Nunc viverra imperdiet enim. Fusce est.</span></li>
+
<li ></li>
                            <li style=" padding-right: 0.3cm;"><span>Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas.
+
</ol>
                          <ol type="i" style=" padding-left: 0.5cm">
+
</li>
                              <li><span>Proin pharetra nonummy pede.</span></li>
+
                              <li><span>Mauris et orci.</span></li>
+
                          </ol>
+
                      </span></li>
+
  
 +
</ol>
 +
<br/>
  
                        </ol></li>
 
 
                    <li>Saccharomyces </li>
 
                </ol>
 
            </li>
 
            <li><a href="#" style="text-decoration: none; font-size: 18px">
 
                <b>Preparation of Chemical Competent Cells </b>&nbsp;<i class="fa fa-chevron-circle-down"></i></a>
 
                <ol  class="protocolhide" style=" padding-left: 0.1cm; list-style-type: none;">
 
                    <li ></li>
 
 
                </ol>
 
            </li>
 
  
  
        </ol>
 
  <br/>
 
  
 
<h2 style="border-bottom: 5px solid #005b04;padding-left: 0.3cm;">Analytics</h2>
 
<h2 style="border-bottom: 5px solid #005b04;padding-left: 0.3cm;">Analytics</h2>

Revision as of 16:12, 7 October 2016

Protocols

General


  1. Polymerase Chain Reaction (PCR) 
    1. Colony-PCR using Alkaline PEG
    2. Colony-PCR with Heatshock
    3. Extended Colony-PCR
  2. Precipitation 
    1. test
  3. Cell Lysis 
    1. Lysozym
      1. Centrifuge the samples for 4 min at 4°C and 14000 rpm.
      2. Freeze the "dried" pellet overnight.
      3. Add 150 µL buffer on the pellet, resuspend it (e.g. with LB-medium) and incubate for 5 min at room temperature.
      4. Add 150 µL lysozym solution (c = 5-8 mg/mL).
      5. Incubate the samples for 1 h at 37°C and 250 rpm.
      6. Centrifuge the samples for 4 min at 4°C and 14000 rpm.
      7. Decant the supernatant.
    2. Sonication
      1. conditions:
      2. time:            3 min
      3. pulse on:    30 s
      4. pulse off:    20 s
      5. amplitude:  50%
    3. Glass beads
      1. Put some small glass beads into your sample tubes.
      2. Vortex your samples (5-10 min at level 2-6), avoid foam on the top of your sample.
      3. Put the used beads into a waste bottle filling with 70% EtOH for.

  4. Transformation 
    1. E. coli
      1. Thaw 100 µL E. coli DH5α or E. coli BL21(DE3) Gold on ice
      2. Add DNA (2-400 ng/µL: 50 ng if Plasmid DNA; 2 µL (~20 ng) ligation mixture; 1-4 µL PLICing-reaction) to the competent cells and swirl gently
      3. Incubate for 15-30 min on ice.
      4. Perform heatshock of the competent cells using a preheated water bath at 42°C for 45s (DH5α, BL21).
      5. Samples were immediately cooled down 5 min on ice.
      6. Fill up the transformation mix to 1 mL with SOC media and incubate at 37°C at 250rpm for 45 min (45-60 min) for recovery of the cells.
      7. Plate cells on LB agar plate with antibiotic respectively and dry them under the clean bench.
        1. 200 µL
        2. resuspended pellet (from centrifugation of the leftover)
      8. Incubate the agar plates at 37°C overnight(16-18 hours).
      9. Count single colonies of each agar plate on the next day.
    2. Sonication
      1. Lorem ipsum dolor sit amet, consectetuer adipiscing elit.
      2. Maecenas porttitor congue massa.
      3. Fusce posuere, magna sed pulvinar ultricies, purus lectus malesuada libero, sit amet commodo magna eros quis urna.
      4. Nunc viverra imperdiet enim. Fusce est.
      5. Nunc viverra imperdiet enim. Fusce est.
      6. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas.
        1. Proin pharetra nonummy pede.
        2. Mauris et orci.
    3. Saccharomyces
  5. Preparation of Chemical Competent Cells  

Analytics


  1. Gelelectrophoresis 
  2. Analysis of Expression Level 
    1. Preparation of pre-culture
      1. Fill 20 mL LB media into a (small) flask, add the corresponding antibiotics.
      2. Inoculate the pre-culture.
      3. Incubate your culture at 37°C, 250 rpm for 16-18 hours.
    2. Preparation of main culture
      1. Fill 200 mL LB media into a 1000 mL flask and add the corresponding antibiotics.
      2. Inoculate the main culture with 2-5 mL pre-culture, as to reach an OD600 of 0.1 in the main culture.
      3. Incubate your main culture at 37°C, 250 rpm till it reaches the OD600 of 1 (2-4 hours).
      4. Take a sample (500µL) out of your main culture and store it at -20°C.
      5. Induce the expression of your main culture by adding a final concentration of each 0.5 / 1 / 1.5 mM IPTG.
      6. Incubate at 37°C, 250 rpm for an hour.
      7. Take a sample hourly afterwards. (as described above)
    3. Analysis
      1. Perform a cell lysis with the samples which were taken and frozen at -20°C.
      2. Perform a Skim Milk Assay with every lysed sample.
      3. Perform a SDS-gel with all lysed samples.

  3. SDS 
  4. Skim Milk Assay 
  5. AAPF (Alanin-Alanin-Prolin-Phenylalanin) Assay 

Cloning


  1. Enzymatic Digestion 
  2. Dephosphorylation 
    1. Dephosphorylation after restriction
      1. Add 1 unit of rSAP for every 1 pmol of DNA ends (about 1 μg of a 3 kb plasmid) after restriction (example: 1 µL for 2000 kb) and according buffer (volume according to concentration of buffer).
      2. Incubate at 37°C for 30–60 minutes.
      3. Stop reaction by heat-inactivation of rSAP at 65°C for 5 minutes.
      4. Store at -20°C.
  3. Ligation 
  4. Site Directed Mutagenesis (SDM) and Site Saturation Mutagenesis (SSM)  

Media


  1. LB Medium 
  2. SC Minimal Medium 
  3. SOC Medium 
  4. M9 Medium 
  5. YEP Medium 

Devices

Kits

  1. Plasmid Isolation 
  2. PCR Clean-up 
  3. DNA Extraction from Agarose Gel 
  4. JET Cloning