Team:StanfordBrownSpelman/Lab Techniques5

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  			<h3><center><a href="https://2014.igem.org/Team:StanfordBrownSpelman/Lab">Ligation &amp; Chemically<br>Competent Transformation</a></h3>
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<h5><center>Getting Started</h5>

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<b>Ligation</b> (adapted from openwetware ligation protocol):
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<br /><b>10 μl Recipe:</b>
<br />30-50 ng vector DNA (<a href="http://www.insilico.uni-duesseldorf.de/Lig_Input.html">A calculator to make life easy</a>)
<br />1μl (10%) 10X T4 DNA ligase buffer
<br />0.5μl (.5%) T4 DNA ligase
<br />Top up w/ qH20 up to 10uL

<br /><br /><b>Procedure</b>
<br />Usually heat inactivation of digests is sufficient; difficult ligations might require a proper cleanup
<br />As often as possible, use isolated inserts and vectors to avoid unwanted ligations
<br />If the reaction needs to be greater than 10μl, adjust amount of 10X ligase buffer and T4 DNA ligase so that they remain at 1% and .5% by volume, respectively
<br />For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.
<br />For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours (alternatively, high concentration T4 DNA Ligase can be used in a 10 minute ligation).
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<b>Chemically Competent Transformation</b>
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<br /><b>Materials:</b>
<br />1 aliquot of competent cells
<br />2-4μl ligation mixture
<br />500μl SOC media
<br /><br />Procedure
<br />Thaw cells at 4°C for 5 minutes
<br />Gently mix in ligation product
<br />Incubate at 4°C for 20 min
<br />Meanwhile, warm SOC media to 37°C
<br />Heat shock at 42°C for 30 sec (45 sec for NEB5-alpha cells)
<br />Return to 4°C for 1 min
<br />Add 500μl pre-heated SOC
<br />Incubate at 37°C for 1hr with shaking
<br />Meanwhile, pre-heat plates to 37°C
<br />Plate, one plate w/ 100μl, one plate w/ 150μl
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