Difference between revisions of "Team:Stony Brook/Experiments"

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<p>260/280 ratio should be near 2
 
<p>260/280 ratio should be near 2
 
</p>
 
</p>
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<h3> Transformation Protocol </h3>
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<p>Thaw competent E. coli cells on ice and pipette 50 ul of cells into a Eppendorf tube</p>
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<p>Add 1-5ul containing 1 pg-100 ng of plasmid DNA and flick tube 4-5 times</p>
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<p>Blank with 1ul of room temperature water</p>
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<p>Place tube on ice for 30 minutes</p>
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<p>Heat shock at 42°C for 30 seconds</p>
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<p>Place tube on ice for 5 minutes</p>
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<p>Add 950 ul room temperature SOC to mixture</p>
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<p>Incubate at 37°C for 60 minutes at 250 rpm</p>
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<p>Spread 50-100ul onto a plate and incubate overnight at 37°C</p>
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<h3>Restriction Enzyme Digest Protocol</h3>
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<a href="#"><img src="https://static.igem.org/mediawiki/2017/e/ea/T--Stony_Brook--Protocol3.jpg" style="text-align: center;width:250px;height:250px;"/></a>
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<p>Make negative controls without restriction enzymes</p>
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<p>Incubate at 37°C  for at least one hour</p>
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<h3>Ligation Protocol</h3>
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<a href="#"><img src="https://static.igem.org/mediawiki/2017/e/e6/T--Stony_Brook--Protocol4.jpg" style="text-align: center;width:250px;height:250px;"/></a>
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<p>Use a 1:3 vector to insert ration</p>
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<p>Add T4 DNA Ligase last</p>
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<p>Pipette the mixture up and down microfuge</p>
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<p>Incubate overnight at 16°C</p>
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<h3>PCR Purification Protocol</h3>
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<p>We used the PCR and Gel Clean-up kit by Enzo Life Sciences. Their PCR Purification protocol can be found on page 7 of their <a href　"http://www.enzolifesciences.com/fileadmin/files/manual/ENZ-GEN100_insert.pdf"product manual.＜／ａ＞</p>
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</section>
 
</section>
 
 

Revision as of 20:57, 29 October 2017

Stony Brook 2017