Team:Cambridge-JIC/Protocol

From 2014.igem.org

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<h3 id="Gibson">Gibson Assembly</h3>  
<h3 id="Gibson">Gibson Assembly</h3>  
<p>
<p>
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We can now combine our DNA fragments into the final circular plasmid.  
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We combine our DNA fragments into the target synthetic plasmid using Gibson Assembly.  
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The Gibson assembly reaction is an isothermal one-pot reaction  
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The Gibson Assembly reaction is an isothermal one-pot reaction  
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containing 3 enzymes that will convert the linear double stranded DNA  
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in which three enzymes convert the linear DNA  
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fragments from the PCR into circular DNA. (For more information see the  
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fragments from PCR into circular DNA. (For more information see the  
synbio.org guide, or Gibson et al., Nature Methods, 2009)  
synbio.org guide, or Gibson et al., Nature Methods, 2009)  
</p>
</p>
<ol>
<ol>
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<li> Pipette 0.5 µl of each DNA fragment into a PCR tube, for a total
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<li> Pipette 0.5 µl of each DNA fragment into a PCR tube (make sure all the liquid is in the bottom of the tube) </li>
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volume of 2 µl (make sure all the liquid is in the bottom of the tube) </li>
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<li> Start the PCR machine running a Gibson protocol (50°C for 1 hour,  
<li> Start the PCR machine running a Gibson protocol (50°C for 1 hour,  
then hold at 4°C) </li>
then hold at 4°C) </li>
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<li> Add 6 µl of 1.33x Gibson Master Mix to the tube </li>
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<li> Add 3n µl of 1.33x Gibson Master Mix to the tube (where n is the total volume of the fragments </li>
<li> Place immediately into a PCR machine </li>
<li> Place immediately into a PCR machine </li>
</ol>  
</ol>  
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adding the master mix. If you wait too long (more than 20-30 secs), the  
adding the master mix. If you wait too long (more than 20-30 secs), the  
exonuclease enzyme (which does not work well at 50°C) will degrade too  
exonuclease enzyme (which does not work well at 50°C) will degrade too  
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much of the DNA, lowering the efficiency of the reaction significantly
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much of the DNA, lowering the efficiency of the reaction significantly.
</p>
</p>
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 +
<p>
 +
Second Note: a PCR tube with water in place of the Master Mix can be used as a negative control. (It should contain 0.5 µl of each DNA fragment and 3n µl of water).
<h3 id="E._coli_Transformation">E. coli Transformation </h3>
<h3 id="E._coli_Transformation">E. coli Transformation </h3>

Revision as of 15:42, 1 August 2014