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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> | ||
Latest revision as of 03:33, 4 October 2012
