Difference between revisions of "Team:Freiburg/Protocols/TerrificBroth"

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<h1 class="sectionedit1">DNA + Cellfree Expression on PDMS</h1>
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<h1 class="sectionedit1">Preparation of Teriffic Broth</h1>
 
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<strong>protocol for spotting of DNA and cellfree expression on PDMS flow cells</strong><br/>
 
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<em>protocol according to AG Roth</em>
 
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<a class="media" href="https://static.igem.org/mediawiki/2015/4/4b/Freiburg_wiki-symbols-orga.png" title="wiki:symbols:orga.png"><img alt="" class="media" src="https://static.igem.org/mediawiki/2015/4/4b/Freiburg_wiki-symbols-orga.png"/></a> material: chemicals, used kits, …<br/>
 
<a class="media" href="https://static.igem.org/mediawiki/2015/4/4b/Freiburg_wiki-symbols-orga.png" title="wiki:symbols:orga.png"><img alt="" class="media" src="https://static.igem.org/mediawiki/2015/4/4b/Freiburg_wiki-symbols-orga.png"/></a> material: chemicals, used kits, …<br/>
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<a class="media" href="https://static.igem.org/mediawiki/2015/b/bb/Freiburg_wiki-symbols-alarm2.png" title="wiki:symbols:alarm2.png"><img alt="" class="media" src="https://static.igem.org/mediawiki/2015/b/bb/Freiburg_wiki-symbols-alarm2.png"/></a> duration: … min<br/>
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<a class="media" href="https://static.igem.org/mediawiki/2015/b/bb/Freiburg_wiki-symbols-alarm2.png" title="wiki:symbols:alarm2.png"><img alt="" class="media" src="https://static.igem.org/mediawiki/2015/b/bb/Freiburg_wiki-symbols-alarm2.png"/></a> duration: 90 min<br/>
 
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Revision as of 21:42, 14 September 2015

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Preparation of Teriffic Broth

material: chemicals, used kits, …
duration: 90 min

Spotting DNA

  1. Spot DNA (c= 25 ng/µl) on flow cell (spot size can be 0.5 µl or more)
  2. Incubate over night at room temperature in humid atmosphere in petri dish sealed with Parafilm
  3. Heat whole petri dish in oven at 60°C for 30min in order to dry the DNA (leave humid tissue inside and make sure the petri dish is still sealed)
  4. put the flow cell in a petri dish filled with H2O and wash it

DNA has bound to surface if water drops appear on it during washing step

  1. dry flow cell with wafergun

Storage of spotted flow cells

  1. store in fridge sealed with parafilm without humid tissue

Cellfree Expression "on air"

  1. Put flow cell with its frontside (with spots) on a clean glass slide
  2. Make sure the flow cell does not collapse!
  3. Add a second glass slide orthogonally to the first one on the backside of the flow cell to give some stability (should look like a cross now)
  4. Prepare 15 µl cellfree Expression mix per flow cell
AG Roth Mix
component concentration volume
H2O 3,75 µl
Buffer 6 µl
Lysate 5,25 µl
  1. Put flow cells in petri dish with humid tissue and sealed with parafilm
  2. Incubate at 37°C
    1. for 25 min (for a quick check)
    2. for 2 h (for full Expression)
  3. Measure fluorescence in microscope of AG Eimer (only possible with Tobi)
,