Team:UIUC-Illinois/Notebook
From 2011.igem.org
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<div class="desc"><img src="https://static.igem.org/mediawiki/2011/6/6a/Uiuc_notebook_2.jpg" /></div> | <div class="desc"><img src="https://static.igem.org/mediawiki/2011/6/6a/Uiuc_notebook_2.jpg" /></div> | ||
+ | <div class="desc">Primers:</div> | ||
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+ | <div class="desc">ATCG <font color="red">T ACTAGT A GCGGCCG CTGCAG</font> CAGTGAATTAATGGCGATGACGC</div> | ||
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+ | <div class="desc">Tm = 68 C</div> | ||
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+ | <div class="desc">GC = 48%</div> | ||
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+ | <div class="desc">ATCG ACTAGTA <font color="green">CTCTAGAAGCGGCCGCGAATTC</font> GCATGCAAGCTTGGCACTGG</div> | ||
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+ | <div class="desc">Tm = 64 C</div> | ||
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+ | <div class="desc">GC = 60%</div> | ||
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+ | <div class="desc">The following PCR rxn was then set up and run with the subsequent parameters:</div> | ||
+ | <div class="desc">0.3 uL pAH125</div> | ||
+ | <div class="desc">2uL primer 1 (100ng/uL)</div> | ||
+ | <div class="desc">2uL primer2 (100ng/uL)</div> | ||
+ | <div class="desc">5uL 10X Pfu buffer (Agilent)</div> | ||
+ | <div class="desc">2.5uL 10mM dNTP mix</div> | ||
+ | <div class="desc">1uL Turbo Polymerase (Agilent)</div> | ||
+ | <div class="desc">37uL ddH2O</div> | ||
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+ | <div class="desc">95C 5 min</div> | ||
+ | <div class="desc">95C 30sec</div> | ||
+ | <div class="desc">59C 30 sec</div> | ||
+ | <div class="desc">72C 3 min</div> | ||
+ | <div class="desc">Cycle 30X</div> | ||
+ | <div class="desc">72C 5 min</div> | ||
+ | <div class="desc">14C forever</div> | ||
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+ | <div class="desc">The pcr rxn was then purified using a Qiagen pcr purification kit. Resulting concentration was 99ng/uL.</div> | ||
+ | <div class="desc">500ng of the reaction was then digested SpeI with the following reaction set up:</div> | ||
+ | <div class="desc">0.5uL pcr product</div> | ||
+ | <div class="desc">5uL NEB Buffer 2</div> | ||
+ | <div class="desc">1uL 100X BSA</div> | ||
+ | <div class="desc">1uL SpeI (from NEB)</div> | ||
+ | <div class="desc">42.5uL ddH2O</div> | ||
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+ | <div class="desc">Digest ran 1 hour 37C and was followed by an 80C heat inactivation.</div> | ||
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+ | <div class="desc">2uL of the digestion reaction was then used in a 20 uL volume ligation reaction. Set up was as follows:</div> | ||
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+ | <div class="desc">2uL SpeI digest</div> | ||
+ | <div class="desc">2uL 10X Promega T4 ligase buffer</div> | ||
+ | <div class="desc">1uL Promega T4 ligase</div> | ||
+ | <div class="desc">15uL ddH2O</div> | ||
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+ | <div class="desc">Ligation reaction ran at for 8 hours at 15C.</div> | ||
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+ | <div class="desc">3uL of the ligation reaction was then transformed into two strains via heat shock as described in our protocol download. The first strain was a normal pir- DH5alpha E. coli strain and the second was a E. coli K-12 pir-116 strain. The latter should allow replication of the plasmid while the first should not. The transformations were plated on LB kan 30ug/mL. Plates were incubated 18 hours at 37C. The following pictures show that robust colonies resulted on the pir-116 transformant plate but not on the pir- DH5alpha plate.</div> | ||
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Revision as of 03:35, 29 September 2011