Team:HokkaidoU Japan/Notebook/Pre experiment/140910 Ptet-B0032-mRFP-dT

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  <h2 class="step-title">Ptet-B0032-mRFP-dT</h2>
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	  <h2 class="step-title">Start</h2>
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PCR
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	        <li>
		  <time>
Thu Sep 4
</time>
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R0040-B0034-E1010-B0015(on pSB6A1) SpeI_PstI_F Tm:72.8&#8451;& Ptet_SpeI_R Tm:72.6&#8451; (vector)
        XbaI_B0032_mRFP_F Tm:76.3&#8451; & PstI_SpeI_R Tm:74.6&#8451; (insert)
        KOD Fx Neo
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Gel extraction
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		  <time>
Thu Sep 4
</time>
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PCR products
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Digestion
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		  <time>
Fri Sep 5
</time>
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	Cut  vector with SpeI, PstI (using 10&times;H buffer) 
        Cut  insert with XbaI, PstI (using 10&times;M buffer)

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Dephosphorylation
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		  <time>
Fri Sep 5
</time>
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Antactic phosphorylate insert (using 10&times;Antarctic phosphatase buffer) 37&#8451;	35min 65&#8451;	10min

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Ligation
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Fri Sep 5
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Ligate vector with insert
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Transformation
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		  <time>
Fri Sep 5
</time>
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	R0040-B0032-E1010-B0015(on pSB1A6)
        5.0&micro;L DNA to DH5&alpha;  

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Colony PCR
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		  <time>
Sat Sep 6
</time>
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R0040-B0032-E1010-B0015(on pSB6A1) EcoI_XbaI_F Tm:70.9&#8451; & RBS_SpeI_R	Tm:59.2&#8451;

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Culture
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	        <li>
		  <time>
Sat Sep 6
</time>
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	      <div class="step-recipe">
R0040-B0032-E1010-B0015(on pSB6A1)
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Mini-Prep
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		  <time>
Sun Sep 7
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R0040-B0032-E1010-B0015(on pSB6A1)
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