Team:UCLA/oliviajessica

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<h1>Core primers</h1>
 
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<p>
 
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Masp1
 
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Overlap 18 long, 54 Tm, 56% GC
 
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Calculated Tm of core “primer”: 62 C, less than 20 long so annealing should be roughly equivalent to Tm
 
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Actual Protocol MaSP 1 Core primers (60.1 by thermosci)
 
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Mix and centrifuge components prior to use
 
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Assembling on ice, add Phusion Polymerase last, transfer to Thermocycler heated to 98 C
 
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      12.8uL Nuclease-free water
 
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4 uL 5X Phusion HF or GC buffer
 
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0.4 uL 10 mM dNTPs
 
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1uL 10 uM Forward Primer
 
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1uL 10uM Reverse Primer
 
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0.6 uL DMSO, (optional, if not used add 0.6 uL water)
 
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0.2 uL Phusion DNA Polymerase
 
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<tr> <td colspan="3"  height="15px"> </td></tr>
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<tr><td colspan="3"> <h3>Protocols</h3></td></tr>
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<th>Step</th><th>Temperature</th><th>Time</th>
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<td width="90%"  valign="top">  
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<p>You should make use of the calendar feature on the wiki and start a lab notebook. This may be looked at by the judges to see how your work progressed throughout the summer. It is a very useful organizational tool as well. </p>
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<td></td>
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<td>Initial Denaturation</td><td>98 C</td><td>30 seconds</td>
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<tr><td><br> <b>PCR for MASP1 and MASP2 </b></br>
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<a href="https://static.igem.org/mediawiki/2014/archive/4/47/20140701232004%21IGEM_PCR_reaction_design.pdf">PCR Protocol</a></td></tr>
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<td><br>35 Cycles</br><br>Denaturation</br><br>Annealing</br><br>Elongation</br> </td><td><br></br><br>98</br><br>60</br><br>72</br></td><td><br></br><br>10 sec</br><br>30 sec</br><br>10 sec</br></td>
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<tr><td><br></br></td></tr>
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<tr><td>Final Extension</td><td>72</td><td>10 minutes</td>
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<tr><td><b>Silk Thread</b></br>
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<a href="https://static.igem.org/mediawiki/2014/6/6e/Silk_Thread_Protocols.pdf">Silk Thread Protocol</a>
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<tr><td>Hold</td><td>4-10</td><td></td></tr>
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<br>Outline</br>
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<ol>
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<li>Construction of the Synthetic Spider Silk Multimeric Gene</li>
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<li>Cloning of the spider silk multimer into the expression vector</li>
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<li>Gene Expression of pETsilk</li>
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<li>E.coli Cell Lysis</li>
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<li>Silk Protein Purification Using IMAC</li>
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<li>Dialysis</li>
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<li>Protein Analysis</li>
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<li>Amino Acid Analysis</li>
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<li>Overall: Timing of Procedure</li>
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<tr><td><h3> Reference </h3><b>Materials Fabrication</b></br>Rockwood, Danielle N., Rucsanda C. Preda, Tuna Yücel, Xiaoqin Wang, Michael L. Lovett, and David L. Kaplan. "Materials Fabrication from Bombyx Mori Silk Fibroin." Nature Protocols 6.10 (2011): 1612-631. Web. <br></br>
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<br><b>Production of MASP1/2</b></br>Teulé, Florence, Alyssa R. Cooper, William A. Furin, Daniela Bittencourt, Elibio L. Rech, Amanda Brooks, and Randolph V. Lewis. "A Protocol for the Production of Recombinant Spider Silk-like Proteins for Artificial Fiber Spinning." Nature Protocols 4.3 (2009): 341-55. Web.</br></td></tr>
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Latest revision as of 22:41, 2 July 2014

iGEM UCLA




Protocols

You should make use of the calendar feature on the wiki and start a lab notebook. This may be looked at by the judges to see how your work progressed throughout the summer. It is a very useful organizational tool as well.


PCR for MASP1 and MASP2
PCR Protocol


Silk Thread
Silk Thread Protocol
Outline
  1. Construction of the Synthetic Spider Silk Multimeric Gene
  2. Cloning of the spider silk multimer into the expression vector
  3. Gene Expression of pETsilk
  4. E.coli Cell Lysis
  5. Silk Protein Purification Using IMAC
  6. Dialysis
  7. Protein Analysis
  8. Amino Acid Analysis
  9. Overall: Timing of Procedure


Reference

Materials Fabrication
Rockwood, Danielle N., Rucsanda C. Preda, Tuna Yücel, Xiaoqin Wang, Michael L. Lovett, and David L. Kaplan. "Materials Fabrication from Bombyx Mori Silk Fibroin." Nature Protocols 6.10 (2011): 1612-631. Web.


Production of MASP1/2
Teulé, Florence, Alyssa R. Cooper, William A. Furin, Daniela Bittencourt, Elibio L. Rech, Amanda Brooks, and Randolph V. Lewis. "A Protocol for the Production of Recombinant Spider Silk-like Proteins for Artificial Fiber Spinning." Nature Protocols 4.3 (2009): 341-55. Web.

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