UTK Knoxville- September

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9/8/12 <br>
Site-directed mutagenesis performed on pLNCX2 vector to correct Neomycin restriction site that conflicts with BioBrick construction.<br><br>

9/9/12<br>
Colonies picked from plates of Neomycin site-directed mutagenesis<br><br>


9/10/12 <br>
Neomycin site-directed mutagenesis cultures were plasmid extracted and check digestions performed to confirm corrected digestion site. Correct plasmids were then used as template for Neomycin PCR.<br><br>

9/11/12<br>
Digestions performed: <br>
J63010 + pIRES + GFP + cyc: Xba and PstI<br>
J63010 +pIRES2 + GFP + cyc: Xba and PstI<br>
J63010 +pSAP + GFP + cyc: Xba and PstI<br>
J63010 +YAP1 + GFP + cyc: Xba and PstI<br>
J63010 +URE2 + GFP + cyc: Xba and PstI<br>
J63010 + HAP4 + GFP + cyc: Xba and PstI<br>
J63010 +Adh + mOrange : SpeI and PstI<br><br>


Ligations performed to create constructs: <br>
J63010 +Adh + mOrange + pIRES + GFP + cyc<br>
J63010 +Adh + mOrange + pIRES2 + GFP + cyc<br>
J63010 +Adh + mOrange + pSAP + GFP + cyc<br>
J63010 +Adh + mOrange + YAP1 + GFP + cyc<br>
J63010 +Adh + mOrange + URE2 + GFP + cyc<br>
J63010 +Adh + mOrange + HAP4 + GFP + cyc<br>
<br>
9/21/12<br>
Colony PCR Confirmed:<br> 
J63010 +Adh + mOrange + pIRES + GFP + cyc<br>
J63010 +Adh + mOrange + pIRES2 + GFP + cyc<br>
J63010 +Adh + mOrange + pSAP + GFP + cyc<br>
<br>
9/23/12<br>
Colony PCR Confirmed: <br>
J63010 + Adh + Neo<br>
J63010 +Adh + mOrange + YAP1 + GFP + cyc<br>
J63010 +Adh + mOrange + URE2 + GFP + cyc<br>
J63010 +Adh + mOrange + HAP4 + GFP + cyc<br><br>


9/24/12<br>
Digestions: <br>
J63010 + pIRES + GFP + cyc : SpeI and PstI<br>
J63010 + pIRES2 + GFP + cyc : SpeI and PstI<br>
J63010 + pSAP + GFP + cyc : SpeI and PstI<br>
J63010 + Adh + Neo : XbaI and PstI<br>
J63010 +Adh + mOrange + pIRES + GFP + cyc + Adh + Neo : EcoRI and PstI<br>
J63010 + Adh + mOrange +pIRES2 + GFP + cyc + Adh + Neo: EcoRI and PstI<br>
J63010 + Adh + mOrange +pSAP + GFP + cyc + Adh + Neo: EcoRI and PstI<br>
J63010 + Adh + mOrange + cyc : SpeI and PstI<br>
J63010 + Adh + GFP + cyc: SpeI and PstI<br><br>


9/25/12<br>
Ligations performed to create constructs: <br>
J63010 + Adh + mOrange + cyc + Adh + Neo<br>
J63010 + Adh + GFP + cyc + Adh + Neo<br>
J63010 + pIRES + GFP + cyc + Adh + Neo<br>
J63010 + pIRES2 + GFP + cyc + Adh + Neo<br>
J63010 + pSAP + GFP + cyc + Adh + Neo<br><br>

9/28/12<br>
Colony PCR Confirmed: <br>
J63010 + Adh + mOrange + cyc + Adh + Neo (AOC)<br>
J63010 + Adh + GFP + cyc + Adh + Neo (AGC)<br>
J63010 + pIRES + GFP + cyc + Adh + Neo (pIRES control)<br>
J63010 + pIRES2 + GFP + cyc + Adh + Neo (pIRES2 control)<br>
J63010 + pSAP + GFP + cyc + Adh + Neo (pSAP control)<br><br>

BY4741 Yeast electroporated with:<br>
AOC, AGC, pIRES control, pIRES2 control, pSAP control,pIRES complete, pIRES2 complete, pSAP complete. <br>
Electroporations were plated onto 200ug/ml G418 YPD plates.<br><br>