UTK Knoxville- October

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Revision as of 03:29, 4 October 2012

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

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

9/10/12
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.

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

Ligations performed to create constructs:
J63010 +Adh + mOrange + pIRES + GFP + cyc
J63010 +Adh + mOrange + pIRES2 + GFP + cyc
J63010 +Adh + mOrange + pSAP + GFP + cyc
J63010 +Adh + mOrange + YAP1 + GFP + cyc
J63010 +Adh + mOrange + URE2 + GFP + cyc
J63010 +Adh + mOrange + HAP4 + GFP + cyc

9/21/12
Colony PCR Confirmed:
J63010 +Adh + mOrange + pIRES + GFP + cyc
J63010 +Adh + mOrange + pIRES2 + GFP + cyc
J63010 +Adh + mOrange + pSAP + GFP + cyc

9/23/12
Colony PCR Confirmed:
J63010 + Adh + Neo
J63010 +Adh + mOrange + YAP1 + GFP + cyc
J63010 +Adh + mOrange + URE2 + GFP + cyc
J63010 +Adh + mOrange + HAP4 + GFP + cyc

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

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

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

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

10/1/12

Colonies finally appeared on plates. Colonies were picked into corresponding 200ug/ml G418 YPD plates. No colonies grew in culture.

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 {{:Utk knoxville calendar template}}
-/21/12
+/8/12 <br>
-Colony PCR Confirmed:
+Site-directed mutagenesis performed on pLNCX2 vector to correct Neomycin restriction site that conflicts with BioBrick construction.<br><br>
-J63010 +Adh + mOrange + pIRES + GFP + cyc
-J63010 +Adh + mOrange + pIRES2 + GFP + cyc
-J63010 +Adh + mOrange + pSAP + GFP + cyc
-/23/12
+/9/12<br>
-Colony PCR Confirmed:
+Colonies picked from plates of Neomycin site-directed mutagenesis<br><br>
-J63010 + Adh + Neo
-J63010 +Adh + mOrange + YAP1 + GFP + cyc
-J63010 +Adh + mOrange + URE2 + GFP + cyc
-J63010 +Adh + mOrange + HAP4 + GFP + cyc
-/24/12
+/10/12 <br>
-Digestions:
+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>
-J63010 + pIRES + GFP + cyc : SpeI and PstI
-J63010 + pIRES2 + GFP + cyc : SpeI and PstI
-J63010 + pSAP + GFP + cyc : SpeI and PstI
-J63010 + Adh + Neo : XbaI and PstI
-J63010 +Adh + mOrange + pIRES + GFP + cyc + Adh + Neo : EcoRI and PstI
-J63010 + Adh + mOrange +pIRES2 + GFP + cyc + Adh + Neo: EcoRI and PstI
-J63010 + Adh + mOrange +pSAP + GFP + cyc + Adh + Neo: EcoRI and PstI
-J63010 + Adh + mOrange + cyc : SpeI and PstI
-J63010 + Adh + GFP + cyc: SpeI and PstI
+/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>
-/25/12
-Ligations performed to create constructs:
-J63010 + Adh + mOrange + cyc + Adh + Neo
-J63010 + Adh + GFP + cyc + Adh + Neo
-J63010 + pIRES + GFP + cyc + Adh + Neo
-J63010 + pIRES2 + GFP + cyc + Adh + Neo
-J63010 + pSAP + GFP + cyc + Adh + Neo
-/28/12
+Ligations performed to create constructs: <br>
-Colony PCR Confirmed:
+J63010 +Adh + mOrange + pIRES + GFP + cyc<br>
-J63010 + Adh + mOrange + cyc + Adh + Neo (AOC)
+J63010 +Adh + mOrange + pIRES2 + GFP + cyc<br>
-J63010 + Adh + GFP + cyc + Adh + Neo (AGC)
+J63010 +Adh + mOrange + pSAP + GFP + cyc<br>
-J63010 + pIRES + GFP + cyc + Adh + Neo (pIRES control)
+J63010 +Adh + mOrange + YAP1 + GFP + cyc<br>
-J63010 + pIRES2 + GFP + cyc + Adh + Neo (pIRES2 control)
+J63010 +Adh + mOrange + URE2 + GFP + cyc<br>
-J63010 + pSAP + GFP + cyc + Adh + Neo (pSAP control)
+J63010 +Adh + mOrange + HAP4 + GFP + cyc<br>
+<br>
+/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>
+/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>
-BY4741 Yeast electroporated with:
-AOC, AGC, pIRES control, pIRES2 control, pSAP control,pIRES complete, pIRES2 complete, pSAP complete.
-Electroporations were plated onto 200ug/ml G418 YPD plates.
-/1/12
+/24/12<br>
-Colonies finally appeared on plates. Colonies were picked into corresponding 200ug/ml G418 YPD plates. No colonies grew in culture.
+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>
+/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>
+/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>
+/1/12<br>
+Colonies finally appeared on plates. Colonies were picked into corresponding 200ug/ml G418 YPD plates. No colonies grew in culture.<br>