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Team:NCTU Formosa/notes
From 2013.igem.org
(Difference between revisions)
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| - | <li class="caldot | + | <li class="caldot green"></li> |
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<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>mini-prep of cultivated AlsS, pSB1K3, pSB1A3, and pSB1C3 E.coli (G2)</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
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<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e2"><p>digestion : [Zif268] ES & [AlsS] XP & [pSB1C3] EP (G2)</p></li> |
| - | <li class=" | + | <li class="green e2"><p>ligation : insert[Zif268] ES & [AlsS] XP/Vector[pSB1C3] EP (G2)</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
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<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e2"><p>Transformation of B0034</p></li> |
| - | <li class=" | + | <li class="green e2"><p>Cultivation of transformed E.coli on LBA plate</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
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| - | <li class=" | + | <li class="green e2"><p>Transformation of Zif268+AlsS+pSB1C3 in DH5-alpha (G2)</p></li> |
| - | <li class=" | + | <li class="green e2"><p>Point mutation HivC (PCR, DPN1 digest, TA cloning, transform) (G2)</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e2"><p>PCR of point mutation HivC (G2)</p></li> |
| - | <li class=" | + | <li class="green e2"><p>PCR of insert fragment [Zif268+AlsS] (G2)</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>electrophoresis of insert fragment [Zif268+AlsS]----NOT OK (G2)</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e2"><p>Single colony isolation from pLac LBA plate, and cultivation of pLac E.coli in liquid LBA</p></li> |
| - | <li class=" | + | <li class="green e2"><p>Single colony isolation from pSB1K3 LBK plate, and cultivation of pSB1K3 E.coli in liquid LBK</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>mini-prep of cultivated pLac & pSB1K3 E.coli</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>digestion : [pSB1K3] EP</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e2"><p>Single colony isolation from Zif268 LBA plate, and cultivation of Zif268 E.coli in liquid LBA</p></li> |
| - | <li class=" | + | <li class="green e2"><p>Single colony isolation from AlsS LBA plate, and cultivation of AlsS E.coli in liquid LBA</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e2"><p>mini-prep of cultivated Zif268 Ecoli & AlsS Ecoli</p></li> |
| - | <li class=" | + | <li class="green e2"><p>digestion :[Zif268] ES/[AlsS] XP</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>ligation : insert Zif268 [ES] & AlsS [XP]/Vector pSB1K3 [EP]</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>transformation of Zif268+AlsS+pSB1K3 and cultivation on LB-K plate</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e2"><p>digestion : [pSB1K3] EP</p></li> |
| - | <li class=" | + | <li class="green e2"><p>digestion : [pSB1K3] EP</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e4"><p>PCR of insert fragment [Zif268+AlsS] OK</p></li> |
| - | <li class=" | + | <li class="green e4"><p>DNA Sequencing OK</p></li> |
| - | <li class=" | + | <li class="green e4"><p>ligation : point mutation HivC</p></li> |
| - | <li class=" | + | <li class="green e4"><p>TA cloning : point mutation HivC </p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>Single colony isolation from Zif268+AlsS LB-K plate, and cultivation of in liquid LB-K</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e3"><p>mini-prep of cultivated Zif268+ AlsS E. coli</p></li> |
| - | <li class=" | + | <li class="green e3"><p>transformation of point mutation HivC and cultivation on LB-A plate</p></li> |
| - | <li class=" | + | <li class="green e3"><p>transformation of B0034 and cultivation on LB-A plate</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e2"><p>Single colony isolation from HivC LB-A plate, and in liquid LB-A</p></li> |
| - | <li class=" | + | <li class="green e2"><p>Single colony isolation from B0034 LB-A plate, and in liquid LB-A</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 541: | Line 541: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>mini-prep of cultivated point mutation HivC E. coli & B0034 E. coli </p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>digestion : Zif268+AlsS [XP]</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>Single colony isolation from ilvD LB-A plate, and cultivation in liquid LB-A </p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e3"><p>mini-prep of cultivated ilvD E. coli</p></li> |
| - | <li class=" | + | <li class="green e3"><p>digestion : ilvD [EP] & pSB1K3 [EP]</p></li> |
| - | <li class=" | + | <li class="green e3"><p>transformation of 37℃ RBS and cultivation on LB-C plate</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>Single colony isolation from HivC LB-A plate, and cultivation in liquid LB-A </p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e4"><p>digestion : B0034 [SP]</p></li> |
| - | <li class=" | + | <li class="green e4"><p>gel extraction</p></li> |
| - | <li class=" | + | <li class="green e4"><p>ligation :insert Zif268+AlsS [XP]/Vector B0034 [SP]</p></li> |
| - | <li class=" | + | <li class="green e4"><p>mini-prep of cultivated Hivc E. coli</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e2"><p>transformation of B0034+Zif268+AlsS and cultivation on LB-A plate</p></li> |
| - | <li class=" | + | <li class="green e2"><p>transformation of HivC and cultivation on LB-A plate</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e8"><p>PCR of insert fragment [B0034+Zif268+AlsS] OK</p></li> |
| - | <li class=" | + | <li class="green e8"><p>DNA Sequencing NOT OK</p></li> |
| - | <li class=" | + | <li class="green e8"><p>digestion : B0034 [SP] & Zif268+AlsS [XP]</p></li> |
| - | <li class=" | + | <li class="green e8"><p>gel extraction</p></li> |
| - | <li class=" | + | <li class="green e8"><p>ligation :insert Zif268+AlsS [XP]/Vector B0034+pSB1A3 [SP]</p></li> |
| - | <li class=" | + | <li class="green e8"><p>transformation of B0034+Zif268+AlsS and cultivation on LB-A plate</p></li> |
| - | <li class=" | + | <li class="green e8"><p>Single colony isolation from B0034 LB-A plate, and cultivation in liquid LB-A</p></li> |
| - | <li class=" | + | <li class="green e8"><p>SCI from ilvD LB-K plate, and cultivation in liquid LB-K</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e4"><p>PCR of insert fragment [B0034+Zif268+AlsS] NOT OK</p></li> |
| - | <li class=" | + | <li class="green e4"><p>transformation of B0034+Zif268+AlsS and cultivation on LB-A plate</p></li> |
| - | <li class=" | + | <li class="green e4"><p>mini-prep of cultivated B0034 and ilvD E. coli</p></li> |
| - | <li class=" | + | <li class="green e4"><p>Single colony isolation from HivC LB-A plate, and cultivation in liquid LB-A</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e2"><p>mini-prep of cultivated HivC E. coli</p></li> |
| - | <li class=" | + | <li class="green e2"><p>digestion : B0034 [SP] & ilvD [XP]</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e3"><p>digestion : pSB1C3 [EP] & HivC [ES] & HivC [SP]</p></li> |
| - | <li class=" | + | <li class="green e3"><p>ligation :insert HivC [ES] & ilvD [XP]/Vector pSB1C3 [EP]</p></li> |
| - | <li class=" | + | <li class="green e3"><p>ligation :insert ilvD [XP]/Vector HivC+pSB1A3 [SP]</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>transformation of HivC+ilvD and cultivation on LB-A & LB-C plate</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 792: | Line 792: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 798: | Line 798: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>PCR of insert fragment [B0034+Zif268+AlsS] NOT OK</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 807: | Line 807: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 815: | Line 815: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e3"><p>transformation of pSB1A3 & pSB1C3 and cultivation on LB-A & LB-C plate</p></li> |
| - | <li class=" | + | <li class="green e3"><p>PCR of insert fragment [HivC+ilvD] OK</p></li> |
| - | <li class=" | + | <li class="green e3"><p>DNA sequencing NOT OK</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 830: | Line 830: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 836: | Line 836: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>Single colony isolation from pSB1C3 LB-C plate, and cultivation in liquid LB-C</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 845: | Line 845: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 851: | Line 851: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>mini-prep of cultivated & pSB1C3 E. coli</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 976: | Line 976: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 984: | Line 984: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e3"><p>digestion : pLac [EP] & pSB1K3 [EP]</p></li> |
| - | <li class=" | + | <li class="green e3"><p>ligation :insert pLac [EP]/Vector pSB1K3 [EP]</p></li> |
| - | <li class=" | + | <li class="green e3"><p>transformation of pLac+pSB1K3 and cultivation on LB-K plate</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 995: | Line 995: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,004: | Line 1,004: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e4"><p>PCR of insert fragment [pLac+pSB1K3] OK</p></li> |
| - | <li class=" | + | <li class="green e4"><p>DNA Sequencing OK</p></li> |
| - | <li class=" | + | <li class="green e4"><p>transformation of B0034_new and cultivation on LB-A plate</p></li> |
| - | <li class=" | + | <li class="green e4"><p>Single colony isolation from B0034 LB-A plate, and cultivation in liquid LB-A</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,016: | Line 1,016: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,026: | Line 1,026: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e5"><p>mini-prep of cultivated B0034 E. coli</p></li> |
| - | <li class=" | + | <li class="green e5"><p>digestion : B0034 [SP] </p></li> |
| - | <li class=" | + | <li class="green e5"><p>ligation :insert Zif268+AlsS [XP]/Vector B0034+pSB1A3 [SP]</p></li> |
| - | <li class=" | + | <li class="green e5"><p>transformation of B0034+Zif268+AlsS</p></li> |
| - | <li class=" | + | <li class="green e5"><p>cultivation on LB-A plate</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,039: | Line 1,039: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,046: | Line 1,046: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e2"><p>PCR of insert fragment [B0034+Zif268+AlsS]-----self ligation</p></li> |
| - | <li class=" | + | <li class="green e2"><p>transformation of 37℃ RBS and cultivation on LB-C plate</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,094: | Line 1,094: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,100: | Line 1,100: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>transformation of 37℃ RBS and cultivation on LB-Kplate</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,109: | Line 1,109: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,115: | Line 1,115: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>Single colony isolation from 37℃ RBS LB-K plate, and cultivation in liquid LB-K</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,124: | Line 1,124: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,131: | Line 1,131: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e2"><p>digestion : B0034 [ES] & pSB1C3 [EP]</p></li> |
| - | <li class=" | + | <li class="green e2"><p>mini-prep of cultivated 37℃ RBS E. coli</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,141: | Line 1,141: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,148: | Line 1,148: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e2"><p>ligation :insert B0034 [ES] & pSB1C3 [EP]/Vector pSB1C3 [EP]</p></li> |
| - | <li class=" | + | <li class="green e2"><p>transformation of B0034+Zif268+AlsS and cultivation on LB-C plate</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,158: | Line 1,158: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,167: | Line 1,167: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e4"><p>digestion : pLac [ES] & pLac [SP]</p></li> |
| - | <li class=" | + | <li class="green e4"><p>digestion : B0034 [SP] Kr (gel extraction) , B0034 [SP] Ar (gel extraction) , 37℃ RBS [XP] & HivC [XP]</p></li> |
| - | <li class=" | + | <li class="green e4"><p>ligation : (1. 2 parts) insert HivC [XP], Vector B0034+pSB1K3 [SP]</p></li> |
| - | <li class=" | + | <li class="green e4"><p>ligation : (2. 3 parts) insert HivC [XP], Vector B0034+pSB1A3 [SP]</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,208: | Line 1,208: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,216: | Line 1,216: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e3"><p>PCR of insert fragment [B0034+Zif268+AlsS] OK</p></li> |
| - | <li class=" | + | <li class="green e3"><p>DNA Sequencing OK</p></li> |
| - | <li class=" | + | <li class="green e3"><p>transformation of B0034+ HivC and cultivation on LB-A & LB-K plate</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,253: | Line 1,253: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,261: | Line 1,261: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e3"><p>Single colony isolation from pSB1A3 LB-A plate, and cultivation in liquid LB-A</p></li> |
| - | <li class=" | + | <li class="green e3"><p>Single colony isolation from B0034+Zif268+AlsS LB-C plate, and cultivation in liquid LB-C</p></li> |
| - | <li class=" | + | <li class="green e3"><p>Single colony isolation from B0034+HivC LB-A & K plate, and cultivation in liquid LB-A & K</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,272: | Line 1,272: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,283: | Line 1,283: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e6"><p>mini-prep of cultivated pSB1A3 E. coli & B0034+Zif268+AlsS</p></li> |
| - | <li class=" | + | <li class="green e6"><p>digestion: pSB1A3 [EP] & B0034+Zif268+AlsS [XP] </p></li> |
| - | <li class=" | + | <li class="green e6"><p>ligation: (1.3 parts)insert pLac [ES] & B0034+Zif268+AlsS [XP], Vector pSB1A3 [EP]</p></li> |
| - | <li class=" | + | <li class="green e6"><p>ligation: (2.2 parts)insert B0034+Zif268+AlsS [XP], Vector pLac+pSB1K3 [SP]</p></li> |
| - | <li class=" | + | <li class="green e6"><p>transformation of pLac+B0034+Zif268+AlsS and cultivation on LB-A plate & LB-K plate</p></li> |
| - | <li class=" | + | <li class="green e6"><p>mini-prep of cultivated B0034+HivC (Ar & Kr)</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,301: | Line 1,301: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,308: | Line 1,308: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e2"><p>PCR of insert fragment [pLac+B0034+Zif268+AlsS] OK</p></li> |
| - | <li class=" | + | <li class="green e2"><p>DNA Sequencing 3 parts OK</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,318: | Line 1,318: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,324: | Line 1,324: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>digestion : 37℃ RBS [XP] , ilvD [ES] & pSB1C3 [EP]</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,346: | Line 1,346: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,353: | Line 1,353: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e2"><p>digestion : HivC+ilvD-3,18 [EP]</p></li> |
| - | <li class=" | + | <li class="green e2"><p>ligation :insert ilvD[ES] & 37℃ RBS [XP], Vector pSB1C3 [EP]</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,363: | Line 1,363: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,369: | Line 1,369: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>Single colony isolation from pLac+B0034+Zif268+AlsS LB-A plate, and cultivation in liquid LB-A</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,378: | Line 1,378: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,385: | Line 1,385: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e2"><p>mini-prep of cultivated pLac+B0034+Zif268+AlsS E. coli</p></li> |
| - | <li class=" | + | <li class="green e2"><p>transformation of ilvD+37℃ RBS and cultivation on LB-C plate</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,395: | Line 1,395: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,402: | Line 1,402: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e2"><p>PCR of insert fragment [ilvD+37℃]-----OK</p></li> |
| - | <li class=" | + | <li class="green e2"><p>DNA Sequencing OK</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,455: | Line 1,455: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,462: | Line 1,462: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e2"><p>Single colony isolation from PBSII+ilvC LB-K plate, and cultivation in liquid LB-K</p></li> |
| - | <li class=" | + | <li class="green e2"><p>ligation : insert HivC [XP], vector B0034+pSB1A3 [SP]</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,472: | Line 1,472: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,482: | Line 1,482: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e5"><p>mini-prep of cultivated PBSII+ilvC E. coli</p></li> |
| - | <li class=" | + | <li class="green e5"><p>digestion : pSB1C3 [EP] & PBSII+ilvC [XP] </p></li> |
| - | <li class=" | + | <li class="green e5"><p>ligation : insert B0034 [ES] & PBSII+ilvC [XP], vector pSB1C3 [EP]</p></li> |
| - | <li class=" | + | <li class="green e5"><p>transformation of B0034+PBSII+ilvC and cultivation on LB-C plate</p></li> |
| - | <li class=" | + | <li class="green e5"><p>transformation of B0034+HivC and cultivation on LB-A plate</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,611: | Line 1,611: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,618: | Line 1,618: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e2"><p>PCR of insert fragment [B0034+PBSII+ilvC] OK!</p></li> |
| - | <li class=" | + | <li class="green e2"><p>DNA Sequencing OK!</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,644: | Line 1,644: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,651: | Line 1,651: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e2"><p>mini-prep of cultivated B0034+PBSII+ilvC E. coli & pLac+B0034+Zif268+AlsS E. coli</p></li> |
</ul> | </ul> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e2"><p>mini-prep of cultivated HivC+ilvD-12,20 E. coli </p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,663: | Line 1,663: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,669: | Line 1,669: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>digestion : HivC+ilvD-12,20 [XP]</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,678: | Line 1,678: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,684: | Line 1,684: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>Design the primer for pLac+B0034+Zif268+AlsS point mutation (pm)</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,693: | Line 1,693: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,702: | Line 1,702: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e4"><p>digestion : B0034 [ES] & HivC [XP] & pSB1C3 [EP] & HivC [EP]</p></li> |
| - | <li class=" | + | <li class="green e4"><p>ligation : insert B0034 [ES] & HivC [XP]/vector pSB1C3 [EP]</p></li> |
| - | <li class=" | + | <li class="green e4"><p>ligation : insert HivC [EP]/vector pSB1C3 [EP]</p></li> |
| - | <li class=" | + | <li class="green e4"><p>transformation of B0034+ HivC & HivC ,and cultivation on LB-C plate</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,714: | Line 1,714: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,720: | Line 1,720: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>PCR of insert fragment [B0034+ HivC] & [HivC] OK</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,729: | Line 1,729: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,736: | Line 1,736: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e2"><p>Design the primer for B0034+PBSII+ilvC point mutation (pm)</p></li> |
| - | <li class=" | + | <li class="green e2"><p>Single colony isolation from HivC, B0034+HivC & ilvD+37℃ RBS LB-C plate, and cultivation in liquid LB-C</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,746: | Line 1,746: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,754: | Line 1,754: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e3"><p>mini-prep of cultivated HivC & B0034+HivC & ilvD+37℃ RBS E. coli </p></li> |
| - | <li class=" | + | <li class="green e3"><p>Testing the temperature of the point mutation between of HivC & ilvD by the m.p 50℃ of PCR</p></li> |
| - | <li class=" | + | <li class="green e3"><p>ligation: insert B0034+HivC [ES] & ilvD [XP]/vector pSB1A3 [EP];insert B0034+HivC [ES] & ilvD+37℃ RBS [XP]/vector pSB1A3 [EP] |
</p></li> | </p></li> | ||
</ul> | </ul> | ||
| Line 1,769: | Line 1,769: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,776: | Line 1,776: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e2"><p>transformation of B0034+ HivC+ ilvD & B0034+ HivC+ ilvD+37℃ RBS ,and cultivation on LB-A plate</p></li> |
| - | <li class=" | + | <li class="green e2"><p>Testing the temperature of the point mutation between of pLac+B0034+Zif268 & AlsS by the m.p 55℃ of PCR failed</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,786: | Line 1,786: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,793: | Line 1,793: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e2"><p>DNA Sequencing B0034+HivC & HivC OK</p></li> |
| - | <li class=" | + | <li class="green e2"><p>PCR of insert fragment [B0034+ HivC+ilvD] & [B0034+ HivC+ilvD+37℃ RBS] OK</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,803: | Line 1,803: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,810: | Line 1,810: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e2"><p>Single colony isolation from B0034+ HivC+ilvD+37℃ RBS & B0034+ HivC+ ilvD LB-A plate, and cultivation in liquid LB-A</p></li> |
| - | <li class=" | + | <li class="green e2"><p>Single colony isolation from HivC(TA) LB-A plate, and cultivation in liquid LB-A</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,820: | Line 1,820: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,827: | Line 1,827: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e2"><p>mini-prep of cultivated B0034+ HivC+ilvD+37℃ RBS , B0034+ HivC+ ilvD & HivC(TA) E. coli </p></li> |
| - | <li class=" | + | <li class="green e2"><p>Testing the temperature of the point mutation between of pLac+B0034+Zif268 & AlsS by the m.p 50 & 52℃ of PCR----50℃ is better</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,837: | Line 1,837: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,843: | Line 1,843: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>PCR of insert fragment [pLac+B0034+Zif268+AlsS] with designed primer for point mutation -----NOT OK</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,852: | Line 1,852: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,858: | Line 1,858: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>DNA Sequencing B0034+ HivC+ilvD+37℃ RBS & B0034+ HivC+ ilvD OK</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,910: | Line 1,910: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,917: | Line 1,917: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e2"><p>PCR of insert fragment [pLac+B0034+Zif268+AlsS] with designed primer for point mutation OK</p></li> |
| - | <li class=" | + | <li class="green e2"><p>digestion: pLac+B0034+Zif268+AlsS [DPn1]</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,927: | Line 1,927: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,933: | Line 1,933: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>transformation of pLac+B0034+Zif268+AlsS (point mutation), and cultivation on LB-A plate</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,942: | Line 1,942: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,948: | Line 1,948: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>Single colony isolation from pLac+B0034+Zif268+AlsS (pm) LB-A plate, and cultivation in liquid LB-A</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,957: | Line 1,957: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,964: | Line 1,964: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e2"><p>mini-prep of cultivated pLac+B0034+Zif268+AlsS (pm) E. coli </p></li> |
| - | <li class=" | + | <li class="green e2"><p>digestion : pLac+B0034+Zif268+AlsS (pm) [EP] </p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,974: | Line 1,974: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 1,980: | Line 1,980: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>DNA sequencing OK</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,006: | Line 2,006: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,013: | Line 2,013: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e2"><p>PCR of insert fragment [B0034+ PBSII+ilvC] with designed primer for point mutation OK</p></li> |
| - | <li class=" | + | <li class="green e2"><p>digestion : B0034+ PBSII+ilvC [DPn1]</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,023: | Line 2,023: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,029: | Line 2,029: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>transformation of B0034+ PBSII+ilvC (pm), and cultivation on LB-C plate------Fail</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,038: | Line 2,038: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,044: | Line 2,044: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>PCR of insert fragment [B0034+ PBSII+ilvC] with designed primer for point mutation OK</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,053: | Line 2,053: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,059: | Line 2,059: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>digestion : B0034+ PBSII+ilvC [DPn1]</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,068: | Line 2,068: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,074: | Line 2,074: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>transformation of B0034+ PBSII+ilvC (pm), and cultivation on LB-C plate</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,083: | Line 2,083: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,090: | Line 2,090: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e2"><p>PCR of insert fragment [B0034+ PBSII+ilvC] (pm) OK</p></li> |
| - | <li class=" | + | <li class="green e2"><p>Single colony isolation from B0034+ PBSII+ilvC (pm) LB-C plate, and cultivation in liquid LB-C</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,125: | Line 2,125: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,132: | Line 2,132: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e2"><p>mini-prep of cultivated B0034+ PBSII+ilvC (pm) E. coli </p></li> |
| - | <li class=" | + | <li class="green e2"><p>digestion : B0034+ PBSII+ilvC (pm) [EP] </p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,142: | Line 2,142: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,148: | Line 2,148: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>DNA sequencing OK</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,209: | Line 2,209: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,215: | Line 2,215: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>digestion : pLac+B0034+zif268+AlsS (pm) [ES] & B0034+ PBSII+ilvC (pm) [XP]</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,236: | Line 2,236: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,245: | Line 2,245: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e4"><p>Testing the temperature of the point mutation between of HivC & ilvD by the m.p 50℃ of PCR</p></li> |
| - | <li class=" | + | <li class="green e4"><p>digestion : pSB1K3 [EP]</p></li> |
| - | <li class=" | + | <li class="green e4"><p>ligation : insert pLac+B0034+zif268+AlsS (pm) [ES] & B0034+ PBSII+ilvC (pm) [XP]/vector pSB1K3 [EP]</p></li> |
| - | <li class=" | + | <li class="green e4"><p>transformation of pLac+B0034+zif268+AlsS+ B0034+ PBSII+ilvC (pm) and cultivation on LB-K plate</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,257: | Line 2,257: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,266: | Line 2,266: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e4"><p>Testing the temperature of the point mutation between of HivC & ilvD by the m.p 48℃ of PCR</p></li> |
| - | <li class=" | + | <li class="green e4"><p>Digestion : B0034+ HivC+ilvD+37℃ RBS [DPn1] </p></li> |
| - | <li class=" | + | <li class="green e4"><p>PCR of insert fragment [pLac+B0034+zif268+AlsS+ B0034+ PBSII+ilvC] (pm) OK</p></li> |
| - | <li class=" | + | <li class="green e4"><p>SCI from pLac+B0034+zif268+AlsS+ B0034+ PBSII+ilvC (pm) LB-K plate, and cultivation in liquid LB-K</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,278: | Line 2,278: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,286: | Line 2,286: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e3"><p>mini-prep of cultivated pLac+B0034+zif268+AlsS+ B0034+ PBSII+ilvC (pm) E. coli </p></li> |
| - | <li class=" | + | <li class="green e3"><p>DNA sequencing OK</p></li> |
| - | <li class=" | + | <li class="green e3"><p>culture condition test: activation DH5αovernight</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,297: | Line 2,297: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,304: | Line 2,304: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e2"><p>transfer to new medium(1/100), OD0.2 start counting culture time</p></li> |
| - | <li class=" | + | <li class="green e2"><p>transfer to 30゜C and 27゜C</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,314: | Line 2,314: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,320: | Line 2,320: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>transfer to 30゜C and 27゜C</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,345: | Line 2,345: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,351: | Line 2,351: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>transformation of B0034+ HivC+ ilvD+37℃ RBS (pm),and cultivation on LB-A plate</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,360: | Line 2,360: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,366: | Line 2,366: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>PCR of insert fragment [B0034+ HivC+ilvD+37℃ RBS](pm)-----NOT OK</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,375: | Line 2,375: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,382: | Line 2,382: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e2"><p>Point mutation by PCR of insert fragment [B0034+ HivC+ilvD+37℃ RBS]</p></li> |
| - | <li class=" | + | <li class="green e2"><p>Digestion: B0034+ HivC+ilvD+37℃ RBS [DPn1] </p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,405: | Line 2,405: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,411: | Line 2,411: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>transformation of B0034+ HivC+ ilvD+37℃ RBS (pm),and cultivation on LB-A,K,C plate (A sucessful)</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,420: | Line 2,420: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,427: | Line 2,427: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e2"><p>PCR of insert fragment [B0034+ HivC+ilvD+37℃ RBS](pm) ---- OK</p></li> |
| - | <li class=" | + | <li class="green e2"><p>Single colony isolation from B0034+ HivC+ilvD+37℃ RBS (pm) LB-A plate, and cultivation in liquid LB-A</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,437: | Line 2,437: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,445: | Line 2,445: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e3"><p>mini-prep of cultivated B0034+ HivC+ilvD+37℃ RBS (pm) E. coli </p></li> |
| - | <li class=" | + | <li class="green e3"><p>Digestion: pLac+B0034+zif268+AlsS+ B0034+ PBSII+ilvC (pm)(G1) [ES] & B0034+ HivC+ilvD+37℃ RBS (pm)(G2) [XP] </p></li> |
| - | <li class=" | + | <li class="green e3"><p>ligation : insert G1[ES] & G2[XP] |
vector pSB1C3[EP] | vector pSB1C3[EP] | ||
</p></li> | </p></li> | ||
| Line 2,462: | Line 2,462: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,468: | Line 2,468: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>transformation of G1+G2,and cultivation on LB- C plate failed</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,477: | Line 2,477: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,483: | Line 2,483: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>ligation : insert G1[ES] & G2[XP]/vector pSB1C3[EP]</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,492: | Line 2,492: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,500: | Line 2,500: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e3"><p>transformation of G1+G2,and cultivation on LB- C plate</p></li> |
| - | <li class=" | + | <li class="green e3"><p>Digestion: G2’(B0034+HIVC+ilvD) [XP]</p></li> |
| - | <li class=" | + | <li class="green e3"><p>ligation : insert G1[ES] & G2‘[XP]/vector pSB1C3[EP]</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,511: | Line 2,511: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,518: | Line 2,518: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e2"><p>PCR of insert fragment [kivD+B0015]</p></li> |
| - | <li class=" | + | <li class="green e2"><p>transformation of G1+G2’, and cultivation on LB- C plate failed</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,528: | Line 2,528: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,535: | Line 2,535: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e2"><p>Single colony isolation from kivD+B0015 LB-C plate, and cultivation in liquid LB-C</p></li> |
| - | <li class=" | + | <li class="green e2"><p>sample and do GC</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,545: | Line 2,545: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,551: | Line 2,551: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>mini-prep of cultivated kivD+B0015 E. coli </p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,577: | Line 2,577: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,584: | Line 2,584: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e2"><p>Point mutation by PCR of insert fragment [B0034+ HivC+ilvD(G2’)]</p></li> |
| - | <li class=" | + | <li class="green e2"><p>Digestion: G2’ [DPn1]</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,594: | Line 2,594: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,600: | Line 2,600: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>transformation of G2’, and cultivation on LB- A plate</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,609: | Line 2,609: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,616: | Line 2,616: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e2"><p>Digestion: kivD+B0015 [EP] (checking bp----failed)</p></li> |
| - | <li class=" | + | <li class="green e2"><p>Single colony isolation from G2’ (pm) LB-A plate, and cultivation in liquid LB-A</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,683: | Line 2,683: | ||
</div> | </div> | ||
<div class="li"><div class="card"> | <div class="li"><div class="card"> | ||
| - | + | <h3>August 2013</h3> | |
<!---------------------------------------- cal starts ----------------------------------------> | <!---------------------------------------- cal starts ----------------------------------------> | ||
<div class="calendar"> | <div class="calendar"> | ||
| Line 2,742: | Line 2,742: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,748: | Line 2,748: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>mini-prep of cultivated G2’ E. coli</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,849: | Line 2,849: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,856: | Line 2,856: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e2"><p>Digestion: G1 [ES] & G2‘ [XP] & pSB1C3 [EP]</p></li> |
| - | <li class=" | + | <li class="green e2"><p>ligation : insert G1[ES] & G2‘[XP]/vector pSB1C3[EP]</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,866: | Line 2,866: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,872: | Line 2,872: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>strain test: activation of different strains overnight</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,881: | Line 2,881: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,887: | Line 2,887: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>transfer to new medium, OD0.2 IPTGinduction, culture in 37゜C</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,899: | Line 2,899: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,906: | Line 2,906: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e2"><p>Single colony isolation from G1+G2’ LB-C plate, and cultivation in liquid LB-C</p></li> |
| - | <li class=" | + | <li class="green e2"><p>transfer to 27゜C</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,916: | Line 2,916: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,922: | Line 2,922: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>mini-prep of cultivated G1+G2’ E. coli </p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,944: | Line 2,944: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,951: | Line 2,951: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e2"><p>Digestion: Ptet+B0032[ES] & GliI+KivD[XP]</p></li> |
| - | <li class=" | + | <li class="green e2"><p>sample and do GC</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,961: | Line 2,961: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,968: | Line 2,968: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e2"><p>ligation : insert Ptet+B0032[ES] & GliI+KivD[XP]/vector pSB1K3[EP]</p></li> |
| - | <li class=" | + | <li class="green e2"><p>Single colony isolation from G1+G2 LB-C plate, and cultivation in liquid LB-C</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,978: | Line 2,978: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,985: | Line 2,985: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e2"><p>transformation of Ptet+B0032+ GliI+KivD, and cultivation on LB- K plate</p></li> |
| - | <li class=" | + | <li class="green e2"><p>mini-prep of cultivated G1+G2 E. coli</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,995: | Line 2,995: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 3,002: | Line 3,002: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e2"><p>PCR of insert fragment [Ptet+B0032+ GliI+KivD]-----OK</p></li> |
| - | <li class=" | + | <li class="green e2"><p>DNA sequencing------NOT OK</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 3,081: | Line 3,081: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 3,087: | Line 3,087: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>carbon source test: activation DH5α overnight</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 3,096: | Line 3,096: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 3,102: | Line 3,102: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>transfer to new medium(1/100), OD0.2 start counting culture time</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 3,115: | Line 3,115: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 3,121: | Line 3,121: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>culturing for 72hours, inject feeding solution per 24hours</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 3,130: | Line 3,130: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 3,136: | Line 3,136: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green"><p>culturing for 72hours, inject feeding solution per 24hours</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 3,145: | Line 3,145: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 3,152: | Line 3,152: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e2"><p>transformation of DNA program, and cultivation on LB- A plate</p></li> |
| - | <li class=" | + | <li class="green e2"><p>culturing for 72hours, inject feeding solution per 24hours</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 3,162: | Line 3,162: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 3,170: | Line 3,170: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e3"><p>ligation : insert Ptet+B0032[ES] & GliI+KivD[XP]/vector pSB1K3[EP]</p></li> |
| - | <li class=" | + | <li class="green e3"><p>Single colony isolation from DNA program LB-C plate, and cultivation in liquid LB-C</p></li> |
| - | <li class=" | + | <li class="green e3"><p>sample & do GC</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 3,181: | Line 3,181: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 3,189: | Line 3,189: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e3"><p>transformation of Ptet+B0032+ GliI+KivD, and cultivation on LB- K plate</p></li> |
| - | <li class=" | + | <li class="green e3"><p>mini-prep of cultivated DNA program E. coli</p></li> |
| - | <li class=" | + | <li class="green e3"><p>Do GC</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 3,200: | Line 3,200: | ||
<div class="dots"> | <div class="dots"> | ||
<ul> | <ul> | ||
| - | <li class="caldot | + | <li class="caldot green"></li> |
| - | <li class="caldot | + | <li class="caldot green"></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 3,207: | Line 3,207: | ||
<div class="open"> | <div class="open"> | ||
<ul> | <ul> | ||
| - | <li class=" | + | <li class="green e2"><p>PCR of insert fragment [Ptet+B0032+ GliI+KivD] OK</p></li> |
| - | <li class=" | + | <li class="green e2"><p>DNA sequencing NOT OK</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
Revision as of 18:38, 13 September 2013
Notes
The experimental log that records down the purpose, method, result and date of each experiment that we have conducted.
About the notes
(tell something if there's anything to say.)
March 2013
Sunday
Monday
Tuesday
Wednesday
Thursday
Friday
Saturday
mini-prep of cultivated AlsS, pSB1K3, pSB1A3, and pSB1C3 E.coli (G2)
digestion : [Zif268] ES & [AlsS] XP & [pSB1C3] EP (G2)
ligation : insert[Zif268] ES & [AlsS] XP/Vector[pSB1C3] EP (G2)
Transformation of B0034
Cultivation of transformed E.coli on LBA plate
Transformation of Zif268+AlsS+pSB1C3 in DH5-alpha (G2)
Point mutation HivC (PCR, DPN1 digest, TA cloning, transform) (G2)
PCR of point mutation HivC (G2)
PCR of insert fragment [Zif268+AlsS] (G2)
electrophoresis of insert fragment [Zif268+AlsS]----NOT OK (G2)
Single colony isolation from pLac LBA plate, and cultivation of pLac E.coli in liquid LBA
Single colony isolation from pSB1K3 LBK plate, and cultivation of pSB1K3 E.coli in liquid LBK
mini-prep of cultivated pLac & pSB1K3 E.coli
digestion : [pSB1K3] EP
Single colony isolation from Zif268 LBA plate, and cultivation of Zif268 E.coli in liquid LBA
Single colony isolation from AlsS LBA plate, and cultivation of AlsS E.coli in liquid LBA
mini-prep of cultivated Zif268 Ecoli & AlsS Ecoli
digestion :[Zif268] ES/[AlsS] XP
April 2013
Sunday
Monday
Tuesday
Wednesday
Thursday
Friday
Saturday
ligation : insert Zif268 [ES] & AlsS [XP]/Vector pSB1K3 [EP]
transformation of Zif268+AlsS+pSB1K3 and cultivation on LB-K plate
digestion : [pSB1K3] EP
digestion : [pSB1K3] EP
PCR of insert fragment [Zif268+AlsS] OK
DNA Sequencing OK
ligation : point mutation HivC
TA cloning : point mutation HivC
Single colony isolation from Zif268+AlsS LB-K plate, and cultivation of in liquid LB-K
mini-prep of cultivated Zif268+ AlsS E. coli
transformation of point mutation HivC and cultivation on LB-A plate
transformation of B0034 and cultivation on LB-A plate
Single colony isolation from HivC LB-A plate, and in liquid LB-A
Single colony isolation from B0034 LB-A plate, and in liquid LB-A
mini-prep of cultivated point mutation HivC E. coli & B0034 E. coli
digestion : Zif268+AlsS [XP]
Single colony isolation from ilvD LB-A plate, and cultivation in liquid LB-A
mini-prep of cultivated ilvD E. coli
digestion : ilvD [EP] & pSB1K3 [EP]
transformation of 37℃ RBS and cultivation on LB-C plate
Single colony isolation from HivC LB-A plate, and cultivation in liquid LB-A
digestion : B0034 [SP]
gel extraction
ligation :insert Zif268+AlsS [XP]/Vector B0034 [SP]
mini-prep of cultivated Hivc E. coli
transformation of B0034+Zif268+AlsS and cultivation on LB-A plate
transformation of HivC and cultivation on LB-A plate
PCR of insert fragment [B0034+Zif268+AlsS] OK
DNA Sequencing NOT OK
digestion : B0034 [SP] & Zif268+AlsS [XP]
gel extraction
ligation :insert Zif268+AlsS [XP]/Vector B0034+pSB1A3 [SP]
transformation of B0034+Zif268+AlsS and cultivation on LB-A plate
Single colony isolation from B0034 LB-A plate, and cultivation in liquid LB-A
SCI from ilvD LB-K plate, and cultivation in liquid LB-K
PCR of insert fragment [B0034+Zif268+AlsS] NOT OK
transformation of B0034+Zif268+AlsS and cultivation on LB-A plate
mini-prep of cultivated B0034 and ilvD E. coli
Single colony isolation from HivC LB-A plate, and cultivation in liquid LB-A
mini-prep of cultivated HivC E. coli
digestion : B0034 [SP] & ilvD [XP]
digestion : pSB1C3 [EP] & HivC [ES] & HivC [SP]
ligation :insert HivC [ES] & ilvD [XP]/Vector pSB1C3 [EP]
ligation :insert ilvD [XP]/Vector HivC+pSB1A3 [SP]
transformation of HivC+ilvD and cultivation on LB-A & LB-C plate
PCR of insert fragment [B0034+Zif268+AlsS] NOT OK
transformation of pSB1A3 & pSB1C3 and cultivation on LB-A & LB-C plate
PCR of insert fragment [HivC+ilvD] OK
DNA sequencing NOT OK
Single colony isolation from pSB1C3 LB-C plate, and cultivation in liquid LB-C
mini-prep of cultivated & pSB1C3 E. coli
May 2013
Sunday
Monday
Tuesday
Wednesday
Thursday
Friday
Saturday
digestion : pLac [EP] & pSB1K3 [EP]
ligation :insert pLac [EP]/Vector pSB1K3 [EP]
transformation of pLac+pSB1K3 and cultivation on LB-K plate
PCR of insert fragment [pLac+pSB1K3] OK
DNA Sequencing OK
transformation of B0034_new and cultivation on LB-A plate
Single colony isolation from B0034 LB-A plate, and cultivation in liquid LB-A
mini-prep of cultivated B0034 E. coli
digestion : B0034 [SP]
ligation :insert Zif268+AlsS [XP]/Vector B0034+pSB1A3 [SP]
transformation of B0034+Zif268+AlsS
cultivation on LB-A plate
PCR of insert fragment [B0034+Zif268+AlsS]-----self ligation
transformation of 37℃ RBS and cultivation on LB-C plate
transformation of 37℃ RBS and cultivation on LB-Kplate
Single colony isolation from 37℃ RBS LB-K plate, and cultivation in liquid LB-K
digestion : B0034 [ES] & pSB1C3 [EP]
mini-prep of cultivated 37℃ RBS E. coli
ligation :insert B0034 [ES] & pSB1C3 [EP]/Vector pSB1C3 [EP]
transformation of B0034+Zif268+AlsS and cultivation on LB-C plate
digestion : pLac [ES] & pLac [SP]
digestion : B0034 [SP] Kr (gel extraction) , B0034 [SP] Ar (gel extraction) , 37℃ RBS [XP] & HivC [XP]
ligation : (1. 2 parts) insert HivC [XP], Vector B0034+pSB1K3 [SP]
ligation : (2. 3 parts) insert HivC [XP], Vector B0034+pSB1A3 [SP]
PCR of insert fragment [B0034+Zif268+AlsS] OK
DNA Sequencing OK
transformation of B0034+ HivC and cultivation on LB-A & LB-K plate
Single colony isolation from pSB1A3 LB-A plate, and cultivation in liquid LB-A
Single colony isolation from B0034+Zif268+AlsS LB-C plate, and cultivation in liquid LB-C
Single colony isolation from B0034+HivC LB-A & K plate, and cultivation in liquid LB-A & K
mini-prep of cultivated pSB1A3 E. coli & B0034+Zif268+AlsS
digestion: pSB1A3 [EP] & B0034+Zif268+AlsS [XP]
ligation: (1.3 parts)insert pLac [ES] & B0034+Zif268+AlsS [XP], Vector pSB1A3 [EP]
ligation: (2.2 parts)insert B0034+Zif268+AlsS [XP], Vector pLac+pSB1K3 [SP]
transformation of pLac+B0034+Zif268+AlsS and cultivation on LB-A plate & LB-K plate
mini-prep of cultivated B0034+HivC (Ar & Kr)
PCR of insert fragment [pLac+B0034+Zif268+AlsS] OK
DNA Sequencing 3 parts OK
digestion : 37℃ RBS [XP] , ilvD [ES] & pSB1C3 [EP]
digestion : HivC+ilvD-3,18 [EP]
ligation :insert ilvD[ES] & 37℃ RBS [XP], Vector pSB1C3 [EP]
Single colony isolation from pLac+B0034+Zif268+AlsS LB-A plate, and cultivation in liquid LB-A
mini-prep of cultivated pLac+B0034+Zif268+AlsS E. coli
transformation of ilvD+37℃ RBS and cultivation on LB-C plate
PCR of insert fragment [ilvD+37℃]-----OK
DNA Sequencing OK
Single colony isolation from PBSII+ilvC LB-K plate, and cultivation in liquid LB-K
ligation : insert HivC [XP], vector B0034+pSB1A3 [SP]
mini-prep of cultivated PBSII+ilvC E. coli
digestion : pSB1C3 [EP] & PBSII+ilvC [XP]
ligation : insert B0034 [ES] & PBSII+ilvC [XP], vector pSB1C3 [EP]
transformation of B0034+PBSII+ilvC and cultivation on LB-C plate
transformation of B0034+HivC and cultivation on LB-A plate
June 2013
Sunday
Monday
Tuesday
Wednesday
Thursday
Friday
Saturday
PCR of insert fragment [B0034+PBSII+ilvC] OK!
DNA Sequencing OK!
mini-prep of cultivated B0034+PBSII+ilvC E. coli & pLac+B0034+Zif268+AlsS E. coli
mini-prep of cultivated HivC+ilvD-12,20 E. coli
digestion : HivC+ilvD-12,20 [XP]
Design the primer for pLac+B0034+Zif268+AlsS point mutation (pm)
digestion : B0034 [ES] & HivC [XP] & pSB1C3 [EP] & HivC [EP]
ligation : insert B0034 [ES] & HivC [XP]/vector pSB1C3 [EP]
ligation : insert HivC [EP]/vector pSB1C3 [EP]
transformation of B0034+ HivC & HivC ,and cultivation on LB-C plate
PCR of insert fragment [B0034+ HivC] & [HivC] OK
Design the primer for B0034+PBSII+ilvC point mutation (pm)
Single colony isolation from HivC, B0034+HivC & ilvD+37℃ RBS LB-C plate, and cultivation in liquid LB-C
mini-prep of cultivated HivC & B0034+HivC & ilvD+37℃ RBS E. coli
Testing the temperature of the point mutation between of HivC & ilvD by the m.p 50℃ of PCR
ligation: insert B0034+HivC [ES] & ilvD [XP]/vector pSB1A3 [EP];insert B0034+HivC [ES] & ilvD+37℃ RBS [XP]/vector pSB1A3 [EP]
transformation of B0034+ HivC+ ilvD & B0034+ HivC+ ilvD+37℃ RBS ,and cultivation on LB-A plate
Testing the temperature of the point mutation between of pLac+B0034+Zif268 & AlsS by the m.p 55℃ of PCR failed
DNA Sequencing B0034+HivC & HivC OK
PCR of insert fragment [B0034+ HivC+ilvD] & [B0034+ HivC+ilvD+37℃ RBS] OK
Single colony isolation from B0034+ HivC+ilvD+37℃ RBS & B0034+ HivC+ ilvD LB-A plate, and cultivation in liquid LB-A
Single colony isolation from HivC(TA) LB-A plate, and cultivation in liquid LB-A
mini-prep of cultivated B0034+ HivC+ilvD+37℃ RBS , B0034+ HivC+ ilvD & HivC(TA) E. coli
Testing the temperature of the point mutation between of pLac+B0034+Zif268 & AlsS by the m.p 50 & 52℃ of PCR----50℃ is better
PCR of insert fragment [pLac+B0034+Zif268+AlsS] with designed primer for point mutation -----NOT OK
DNA Sequencing B0034+ HivC+ilvD+37℃ RBS & B0034+ HivC+ ilvD OK
PCR of insert fragment [pLac+B0034+Zif268+AlsS] with designed primer for point mutation OK
digestion: pLac+B0034+Zif268+AlsS [DPn1]
transformation of pLac+B0034+Zif268+AlsS (point mutation), and cultivation on LB-A plate
Single colony isolation from pLac+B0034+Zif268+AlsS (pm) LB-A plate, and cultivation in liquid LB-A
mini-prep of cultivated pLac+B0034+Zif268+AlsS (pm) E. coli
digestion : pLac+B0034+Zif268+AlsS (pm) [EP]
DNA sequencing OK
PCR of insert fragment [B0034+ PBSII+ilvC] with designed primer for point mutation OK
digestion : B0034+ PBSII+ilvC [DPn1]
transformation of B0034+ PBSII+ilvC (pm), and cultivation on LB-C plate------Fail
PCR of insert fragment [B0034+ PBSII+ilvC] with designed primer for point mutation OK
digestion : B0034+ PBSII+ilvC [DPn1]
transformation of B0034+ PBSII+ilvC (pm), and cultivation on LB-C plate
PCR of insert fragment [B0034+ PBSII+ilvC] (pm) OK
Single colony isolation from B0034+ PBSII+ilvC (pm) LB-C plate, and cultivation in liquid LB-C
July 2013
Sunday
Monday
Tuesday
Wednesday
Thursday
Friday
Saturday
mini-prep of cultivated B0034+ PBSII+ilvC (pm) E. coli
digestion : B0034+ PBSII+ilvC (pm) [EP]
DNA sequencing OK
digestion : pLac+B0034+zif268+AlsS (pm) [ES] & B0034+ PBSII+ilvC (pm) [XP]
Testing the temperature of the point mutation between of HivC & ilvD by the m.p 50℃ of PCR
digestion : pSB1K3 [EP]
ligation : insert pLac+B0034+zif268+AlsS (pm) [ES] & B0034+ PBSII+ilvC (pm) [XP]/vector pSB1K3 [EP]
transformation of pLac+B0034+zif268+AlsS+ B0034+ PBSII+ilvC (pm) and cultivation on LB-K plate
Testing the temperature of the point mutation between of HivC & ilvD by the m.p 48℃ of PCR
Digestion : B0034+ HivC+ilvD+37℃ RBS [DPn1]
PCR of insert fragment [pLac+B0034+zif268+AlsS+ B0034+ PBSII+ilvC] (pm) OK
SCI from pLac+B0034+zif268+AlsS+ B0034+ PBSII+ilvC (pm) LB-K plate, and cultivation in liquid LB-K
mini-prep of cultivated pLac+B0034+zif268+AlsS+ B0034+ PBSII+ilvC (pm) E. coli
DNA sequencing OK
culture condition test: activation DH5αovernight
transfer to new medium(1/100), OD0.2 start counting culture time
transfer to 30゜C and 27゜C
transfer to 30゜C and 27゜C
transformation of B0034+ HivC+ ilvD+37℃ RBS (pm),and cultivation on LB-A plate
PCR of insert fragment [B0034+ HivC+ilvD+37℃ RBS](pm)-----NOT OK
Point mutation by PCR of insert fragment [B0034+ HivC+ilvD+37℃ RBS]
Digestion: B0034+ HivC+ilvD+37℃ RBS [DPn1]
transformation of B0034+ HivC+ ilvD+37℃ RBS (pm),and cultivation on LB-A,K,C plate (A sucessful)
PCR of insert fragment [B0034+ HivC+ilvD+37℃ RBS](pm) ---- OK
Single colony isolation from B0034+ HivC+ilvD+37℃ RBS (pm) LB-A plate, and cultivation in liquid LB-A
mini-prep of cultivated B0034+ HivC+ilvD+37℃ RBS (pm) E. coli
Digestion: pLac+B0034+zif268+AlsS+ B0034+ PBSII+ilvC (pm)(G1) [ES] & B0034+ HivC+ilvD+37℃ RBS (pm)(G2) [XP]
ligation : insert G1[ES] & G2[XP] vector pSB1C3[EP]
transformation of G1+G2,and cultivation on LB- C plate failed
ligation : insert G1[ES] & G2[XP]/vector pSB1C3[EP]
transformation of G1+G2,and cultivation on LB- C plate
Digestion: G2’(B0034+HIVC+ilvD) [XP]
ligation : insert G1[ES] & G2‘[XP]/vector pSB1C3[EP]
PCR of insert fragment [kivD+B0015]
transformation of G1+G2’, and cultivation on LB- C plate failed
Single colony isolation from kivD+B0015 LB-C plate, and cultivation in liquid LB-C
sample and do GC
mini-prep of cultivated kivD+B0015 E. coli
Point mutation by PCR of insert fragment [B0034+ HivC+ilvD(G2’)]
Digestion: G2’ [DPn1]
transformation of G2’, and cultivation on LB- A plate
Digestion: kivD+B0015 [EP] (checking bp----failed)
Single colony isolation from G2’ (pm) LB-A plate, and cultivation in liquid LB-A
August 2013
Sunday
Monday
Tuesday
Wednesday
Thursday
Friday
Saturday
mini-prep of cultivated G2’ E. coli
Digestion: G1 [ES] & G2‘ [XP] & pSB1C3 [EP]
ligation : insert G1[ES] & G2‘[XP]/vector pSB1C3[EP]
strain test: activation of different strains overnight
transfer to new medium, OD0.2 IPTGinduction, culture in 37゜C
Single colony isolation from G1+G2’ LB-C plate, and cultivation in liquid LB-C
transfer to 27゜C
mini-prep of cultivated G1+G2’ E. coli
Digestion: Ptet+B0032[ES] & GliI+KivD[XP]
sample and do GC
ligation : insert Ptet+B0032[ES] & GliI+KivD[XP]/vector pSB1K3[EP]
Single colony isolation from G1+G2 LB-C plate, and cultivation in liquid LB-C
transformation of Ptet+B0032+ GliI+KivD, and cultivation on LB- K plate
mini-prep of cultivated G1+G2 E. coli
PCR of insert fragment [Ptet+B0032+ GliI+KivD]-----OK
DNA sequencing------NOT OK
carbon source test: activation DH5α overnight
transfer to new medium(1/100), OD0.2 start counting culture time
culturing for 72hours, inject feeding solution per 24hours
culturing for 72hours, inject feeding solution per 24hours
transformation of DNA program, and cultivation on LB- A plate
culturing for 72hours, inject feeding solution per 24hours
ligation : insert Ptet+B0032[ES] & GliI+KivD[XP]/vector pSB1K3[EP]
Single colony isolation from DNA program LB-C plate, and cultivation in liquid LB-C
sample & do GC
transformation of Ptet+B0032+ GliI+KivD, and cultivation on LB- K plate
mini-prep of cultivated DNA program E. coli
Do GC
PCR of insert fragment [Ptet+B0032+ GliI+KivD] OK
DNA sequencing NOT OK
