Difference between revisions of "Team:NTU-LIHPAO-Taiwan/Notebook"

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Transformation
 
Transformation
 
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<Green>PYY part</Green>
 
<Green>PYY part</Green>
 
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Gel electrophoresis:
 
Gel electrophoresis:
 
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Result:
 
Result:
 
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[TAT-PYY]----- Failed(Confirmed it was pollution)
 
[TAT-PYY]----- Failed(Confirmed it was pollution)
 
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[Cll] ----- Succeeded
 
[Cll] ----- Succeeded
 
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Revision as of 17:20, 15 September 2015

NTU-LIHPAO-Taiwan

7/4
Manufacture CP+ plate
7/6
Learn how to transform
Suicide Part:
Transformation of:
[RBS] (BBa_B0034), [C2] (BBa_C0053)
7/7
Learn how to manufacture competent cell
Manufacture competent cell
PYY part:
Transformation of:
[promoter] (BBa_J23100), [signal peptide] (BBa_K554002), [terminator] (BBa_B0015)
Suicide Part:
Transformation of:
[pC1] (BBa_R0051), [pC2] (BBa_R0053), [endolysin] (BBa_K112806), [C1] (BBa_C0051)
7/8
Learn how to extract the plasmid
Learn how to conduct the gel extraction and electrophoresis
Suicide Part:
Extract the plasmid:
[RBS] (BBa_B0034), [C2] (BBa_C0053)
Restriction enzyme:
[RBS] cleave by EcoRI, [C2] cleave by EcoRI and PstI
Gel electrophoresis:
[RBS] ----- Result: Failed (Smeared band was too blurred)
[C2] ----- Result: Succeeded
Transformation of:
[pC1] (BBa_R0051), [pC2] (BBa_R0053), [endolysin] (BBa_K112806) again
7/9
Suicide Part:
Extract the plasmid:
[RBS] (BBa_B0034), [C1] (BBa_C0051), [endolysin] (BBa_K112806)
Gel electrophoresis:
[RBS] ----- Result: Failed (Smeared band was too blurred)
[C1] ----- Result: Succeeded
[endolysin] ----- Result: Failed (Smeared band was too blurred)
PYY part:
Extract the plasmid:
[signal peptide] (BBa_K554002), [terminator] (BBa_B0015)
Gel electrophoresis:
[signal peptide] ----- Result: Succeeded
[terminator] ----- Result: Succeeded
7/10
Manufacture CP+ plate
Manufacture Amp+ plate
Suicide Part:
Gel electrophoresis:
[RBS] (BBa_B0034) ----- Result: Failed (Smeared band was too blurred)
[C2] (BBa_C0053) ----- Result: Succeeded
Gel extraction:
[C2]
7/11
Suicide Part:
Extract the plasmid:
[RBS] (BBa_B0034), [C1] (BBa_C0051), [endolysin] (BBa_K112806)
PYY part:
Transformation of:
[promoter] (BBa_J23101)
7/12
Suicide Part:
Extract the plasmid:
[pC1] (BBa_R0051)
7/13
PYY part:
Extract the plasmid:
[promoter] (BBa_J23101)
Gel electrophoresis (two white colonies & two red colonies):
[promoter] ------ Result: Failed (white colonies didn’t have band, red colonies had band but not the correct size, we speculated that was pollution)
7/20
Discuss about total synthesis
7/13
PYY part:
Transformation of:
[terminator] (BBa_B0015)
7/24
Discuss our project with professor
PYY part:
Extract the plasmid:
[terminator] (BBa_B0015)
Gel electrophoresis:
[terminator] ----- Result: Failed
7/25
Discuss our problems with Instructor
7/26
PYY part:
Cultured [terminator] again
7/27
PYY part:
Extract the plasmid:
[terminator] (BBa_B0015)
Gel electrophoresis:
[terminator] ----- Result: Succeeded
8/2
Suicide Part:
Extract the plasmid:
[RFP](BBa_E1010)
Restriction enzyme:
[RFP] cleave by EcoRI and SpeI
[Cl] cleave by XbaI and PstI
[pSB1A3] cleave by EcoRI and PstI
8/5
Passaged L.c.393
Manufacture MRS plate
PYY part
Redissoluted DNA of total synthesis
Restriction enzyme:
[signalpeptide -PYY] cleave by EcoRI and PstI
Ligation:
[signalpeptide-PYY]+ [pSB1C3]
Transformation
8/6
Passaged L.c.393
PYY part
Restriction enzyme:
[PYY] cleave by EcoRI and PstI
[TAT-PYY] cleave by EcoRI and PstI
[Y2R] cleave by KpnI and XhoI
Ligation:
[PYY]+ [pSB1C3]
[TAT-PYY]+ [pSB1C3]
[Y2R]+[pcDNA]
Transformation
8/7
PYY part
Gel electrophoresis:
Result:
signalpeptide-PYY] ----- Failed (Smeared band was too blurred)
[PYY] ----- Failed (Smeared band was too blurred)
Transform again
8/10
Manufacture CP+ plate
Suicide part
Gel electrophoresis:
Result:
[RFP+Terminator] ----- Failed (Confirmed it was pollution)
[Cl+Terminator] ----- Failed
Clean up:
[RFP+Terminator]
PYY part
Clean up:
[signalpeptide-PYY]+ [pSB1A3]
[PYY]+ [pSB1A3]
[TAT-PYY]+ [pSB1A3]
[Y2R]+[pcDNA]
Transformation
8/11
Suicide part
Clean up:
[Cl+Terminator]
Gel electrophoresis
Result:
[RFP+Terminator]----- Failed (Smeared band was too blurred)
[Cl+Terminator] ----- Failed (Smeared band was too blurred)
PYY part
Extract the plasmid:
[TAT-PYY], [Y2R]
8/12
Suicide part
Redissoluted DNA of total synthesis
Restriction enzyme:
[Lac-RBS] cleave by EcoRI and PstI
[NucA-Terminator] cleave by EcoRI and PstI
[NIsI] cleave by EcoRI and PstI
[LcSPddhol] cleave by EcoRI and PstI
Ligation:
[Lac-RBS]+ [pSB1C3]
[NucA-Terminator]+ [pSB1C3]
[NIsI]+ [pSB1C3]
[LcSPddhol]+ [pSB1C3]
Transformation
PYY part
Gel electrophoresis:
Result:
[TAT-PYY]----- Failed(Confirmed it was pollution)
[Cll] ----- Succeeded
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