Team:UIUC-Illinois/Notebook
From 2011.igem.org
(Difference between revisions)
Line 145: | Line 145: | ||
<div class="desc">The restriction profiles of submitted parts K617003 and 004 are also shown on the same gel above (EcoRI, PstI digested).</div> | <div class="desc">The restriction profiles of submitted parts K617003 and 004 are also shown on the same gel above (EcoRI, PstI digested).</div> | ||
- | <div class="desc"></div> | + | <div class="title">Plasmid Integration Protocol (for CRIM plasmids)</div> |
+ | <div class="desc">K617003 and K617004 Creation</div> | ||
- | <div class="desc"></div> | + | <br><br> |
+ | <div class="desc">Set up pcr reactions using the pAH125 template miniprep that was also used for the creation of the K617000 plasmid. Concentration is 30ng/uL</div> | ||
+ | |||
+ | <br><br> | ||
+ | <div class="desc">Lambda attP P’OP pcr (K617004)</div> | ||
+ | |||
+ | <br><br> | ||
+ | <div class="desc">Primer 1</div> | ||
+ | <div class="desc">ATCG GAATTC GCGGCCGC T TCTAGA G CCATGGCATCACAGTATCGTG</div> | ||
+ | <div class="desc">Tm = 64 C (eq)</div> | ||
+ | <div class="desc">Primer 2</div> | ||
+ | <div class="desc">ATCG CTGCAGCGGCCGCTACTAGTA AGCTTTGCACTGGATTGCGAG</div> | ||
+ | <div class="desc">Tm = 64 C (eq)</div> | ||
+ | <div class="desc">GC=52% </div> | ||
+ | |||
+ | <br><br> | ||
+ | <div class="desc">0.3uL 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</div> | ||
+ | <div class="desc">2.5uL 10mM dNTP mix</div> | ||
+ | <div class="desc">1uL Turbo Polymerase</div> | ||
+ | <div class="desc">37uL ddH2O</div> | ||
+ | |||
+ | <br><br> | ||
+ | <div class="desc">95C 5 min</div> | ||
+ | <div class="desc">95C 30 sec</div> | ||
+ | <div class="desc">59C 30 sec</div> | ||
+ | <div class="desc">72C 1 min</div> | ||
+ | <div class="desc">Cycle 30X</div> | ||
+ | <div class="desc">72C 5 min</div> | ||
+ | <div class="desc">4C forever</div> | ||
+ | |||
+ | <br><br> | ||
+ | <div class="desc">R6K origin pcr (K617003)</div> | ||
+ | |||
+ | <br><br> | ||
+ | <div class="desc">Primer 1’</div> | ||
+ | |||
+ | <br><br> | ||
+ | <div class="desc">ATCG GAATTC GCGGCCGC T TCTAGA G CCATGTCAGCCGTTAAGTGTTCC</div> | ||
+ | <div class="desc">Tm(eq)= 70 C</div> | ||
+ | <div class="desc">GC = 52%</div> | ||
+ | |||
+ | |||
+ | <br><br> | ||
+ | <div class="desc">Primer2’</div> | ||
+ | <div class="desc">ATCG CTGCAGCGGCCGCTACTAGTA GATCTGAAGATCAGCAGTTCAACC</div> | ||
+ | <div class="desc">Tm(eq)= 70 C</div> | ||
+ | <div class="desc">GC = 46%</div> | ||
+ | |||
+ | <br><br> | ||
+ | <div class="desc">0.3uL 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</div></div> | ||
+ | <div class="desc">2.5uL 10mM dNTP mix</div> | ||
+ | <div class="desc">1uL Turbo Polymerase</div> | ||
+ | <div class="desc">37uL ddH2O</div> | ||
+ | |||
+ | <br><br> | ||
+ | <div class="desc">95C 5 min</div> | ||
+ | <div class="desc">95C 30 sec</div> | ||
+ | <div class="desc">65C 30 sec</div> | ||
+ | <div class="desc">72C 1 min</div> | ||
+ | <div class="desc">Cycle 30X</div> | ||
+ | <div class="desc">72C 5 min</div> | ||
+ | <div class="desc">4C forever</div> | ||
+ | |||
+ | <br><br> | ||
+ | <div class="desc">Both pcr reactions were pcr purified using Qiagen pcr purification kits. Both concentrations were approximately 90 ng/uL. </div> | ||
+ | |||
+ | <br><br> | ||
+ | <div class="desc">Products were then digested EcoRI PstI and assembled with pSB1C3 also cut EcoRI PstI to release an RFP generator. Ligation reactions were performed as previously described (between pSB1C3 and the digested pcr fragments). LB Chloramphenicol 25ug/mL were used for selection as well as red white selection as seen in pictures below. </div> | ||
+ | |||
+ | <div class="desc"><img src="https://static.igem.org/mediawiki/2011/a/af/Uiuc_notebook_7.jpg" /></div> | ||
+ | |||
+ | <div class="desc">The three section gel previsouly shown confirms the correct canidates were picted for K617003 and 004.</div> | ||
<div class="desc"></div> | <div class="desc"></div> |
Revision as of 03:51, 29 September 2011