Team:UCLA/oliviajessica

From 2014.igem.org

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<h1>Core primers</h1>  
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<h1> Protocols </h1>
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Masp1
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<br> <b>PCR Protocol for MASP1 and MASP2 </b></br>
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Overlap 18 long, 54 Tm, 56% GC
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<a href="https://static.igem.org/mediawiki/2014/archive/4/47/20140701232004%21IGEM_PCR_reaction_design.pdf">PCR Protocol</a>
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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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<th>Step</th><th>Temperature</th><th>Time</th>
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<td>Initial Denaturation</td><td>98 C</td><td>30 seconds</td>
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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>Final Extension</td><td>72</td><td>10 minutes</td>
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<tr><td>Hold</td><td>4-10</td><td></td></tr>
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Revision as of 23:30, 1 July 2014

Protocols


PCR Protocol for MASP1 and MASP2
PCR Protocol

Retrieved from "http://2014.igem.org/Team:UCLA/oliviajessica"