Team:Warsaw/Journal

From 2013.igem.org

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====1st week: 2.07.13 – 5.07.13====
====1st week: 2.07.13 – 5.07.13====
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Preparation of competent cells for chemical-based transfection. Failure.  
Preparation of competent cells for chemical-based transfection. Failure.  
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====2nd week: 8.07.13 – 12.07.13====
====2nd week: 8.07.13 – 12.07.13====
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Succeed preparation of competent cells for chemical-based transfection. Verification with iGEM’s transformation kit.  
Succeed preparation of competent cells for chemical-based transfection. Verification with iGEM’s transformation kit.  
Preparation of competent cells for electroporation. Failure.  
Preparation of competent cells for electroporation. Failure.  
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====3rd week: 15.07.13 – 19.07.13====
====3rd week: 15.07.13 – 19.07.13====
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Chemical-based transformation. BioBricks: J06504, J23100, B0034, I712074, E0040, B0015, B0017, E2020, E2030. Failure: E2020. Succeed: J06504, J23100, B0034, I712074, E0040, B0015, B0017, E2030.  
Chemical-based transformation. BioBricks: J06504, J23100, B0034, I712074, E0040, B0015, B0017, E2020, E2030. Failure: E2020. Succeed: J06504, J23100, B0034, I712074, E0040, B0015, B0017, E2030.  
Isolate plasmid DNA by alkaline lysis. Quality cheked with spectrophotomeric analysis with NanoDrop.
Isolate plasmid DNA by alkaline lysis. Quality cheked with spectrophotomeric analysis with NanoDrop.
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====4th week: 22.07.13 – 26.07.13====
====4th week: 22.07.13 – 26.07.13====
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Transformation with isolated pladmids and BioBricks: I74609 and K864100. Isolate plasmid DNA (from I73609 and K864100) with mini-prep kit. Digest and ligation of construct: J23100 and B0034 on plasmid pSB1C3.  
Transformation with isolated pladmids and BioBricks: I74609 and K864100. Isolate plasmid DNA (from I73609 and K864100) with mini-prep kit. Digest and ligation of construct: J23100 and B0034 on plasmid pSB1C3.  
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====5th week: 29.07.13 – 1.08.13====
====5th week: 29.07.13 – 1.08.13====
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Preparing construct: J23100 + B0034 + K864100 on plasmid pSB1A3. Measure glowing fluorescent proteins: sfGTP, sfYFP, sfCFP, sfBFP and SYFP2. Superfolded (sf) forms are made by Warsaw Team. SYFP2 is from parts registry.
Preparing construct: J23100 + B0034 + K864100 on plasmid pSB1A3. Measure glowing fluorescent proteins: sfGTP, sfYFP, sfCFP, sfBFP and SYFP2. Superfolded (sf) forms are made by Warsaw Team. SYFP2 is from parts registry.
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====6th week: 5.08.13 – 9.08.13=====
====6th week: 5.08.13 – 9.08.13=====
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Measure glowing fluorescent proteins. Banking strains with our fluorescent proteins.
Measure glowing fluorescent proteins. Banking strains with our fluorescent proteins.
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====7th week: 12.08.13 – 14.08.13====
====7th week: 12.08.13 – 14.08.13====
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Transformation BioBrick: E2050.  
Transformation BioBrick: E2050.  
PCR: sfBFP N-terminal, sfYFP N-terminal and mCherry (J06504) C-terminal (succeed). Isolate plasmid DNA with: sfGFP (I73609), mOrange (E2050), sfCFP, sfYFP and sfBFP.
PCR: sfBFP N-terminal, sfYFP N-terminal and mCherry (J06504) C-terminal (succeed). Isolate plasmid DNA with: sfGFP (I73609), mOrange (E2050), sfCFP, sfYFP and sfBFP.
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====8th week: 19.08.13 – 23.08.13====
====8th week: 19.08.13 – 23.08.13====
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PCR: sfGFP N-terminal (failure), sfCFP N-terminal (succeed) and mCherry N-terminal (succeed).
PCR: sfGFP N-terminal (failure), sfCFP N-terminal (succeed) and mCherry N-terminal (succeed).
Gel-out: sfYFP N-terminal, sfBFP N-terminal and mCherry C-terminal.
Gel-out: sfYFP N-terminal, sfBFP N-terminal and mCherry C-terminal.
Cloning sfCFP N-terminal, mCherry N-terminal, sfYFP N-terminal, sfBFP N-terminal and mCherry C-terminal to pSB1C3 plasmid and inserting it to bacteria.
Cloning sfCFP N-terminal, mCherry N-terminal, sfYFP N-terminal, sfBFP N-terminal and mCherry C-terminal to pSB1C3 plasmid and inserting it to bacteria.
Preparing construct: J23100+B0034+E0040 – failure.
Preparing construct: J23100+B0034+E0040 – failure.
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====9th week: 26.08.13 – 30.08.13====
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'''Verification cloning'''
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====9th week: 26.08.13 – 30.08.13====
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[[File:Klonowanie.jpg|600px|border|caption]]
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<gallery widths=600px heights=800px perrow=1 caption="
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Marker - GeneRuler 100 bp DNA Ladder, ready-to-use (Thermo); sfYFP N 1,2,3,R1,R2,R3 - random clones of sfYFP N-terminal; sfCFP N 1,2,3,R1,R2,R3 - random clones of sfCFP N-terminal; mCherry N 1,2,3,R1,R2,R3 - random clones of mCherry N-terminal; mCherry C 1,2,3,R1,R2,R3 - random clones of mCherry C-terminal; sfBFP N 1,2,3,R1,R2,R3 - random clones of sfBFP N-terminal. <u>Underlined</u> - correct clones.
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Verification cloning.">
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File:Klonowanie.jpg|'''Results'''. Marker - GeneRuler 100 bp DNA Ladder, ready-to-use (Thermo); sfYFP N 1,2,3,R1,R2,R3 - random clones of sfYFP N-terminal; sfCFP N 1,2,3,R1,R2,R3 - random clones of sfCFP N-terminal; mCherry N 1,2,3,R1,R2,R3 - random clones of mCherry N-terminal; mCherry C 1,2,3,R1,R2,R3 - random clones of mCherry C-terminal; sfBFP N 1,2,3,R1,R2,R3 - random clones of sfBFP N-terminal. <u>Underlined</u> - correct clones.
+
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</gallery>
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And great success!  
And great success!  
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PCR: sfGFP N-terminal and GFP C-terminal (various attenuation template DNA) and sfC-terminal (to versions: m6 and m12 to all of superfolded fluorescent proteins). Cloning and inserting it to bacteria.
PCR: sfGFP N-terminal and GFP C-terminal (various attenuation template DNA) and sfC-terminal (to versions: m6 and m12 to all of superfolded fluorescent proteins). Cloning and inserting it to bacteria.
Once again preparing construct: J23100+B0034+E0040.
Once again preparing construct: J23100+B0034+E0040.
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====10th week: 2.09.13 – 7.09.13====
====10th week: 2.09.13 – 7.09.13====
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Welcome school!
Welcome school!
Verification cloning. Unfortunately epic fail.  
Verification cloning. Unfortunately epic fail.  
PCR: sfGFP N-terminal, GFP N-terminal and GFP C-terminal. At least success!  
PCR: sfGFP N-terminal, GFP N-terminal and GFP C-terminal. At least success!  
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====11th week: 10.09.13 – 14.09.13====
====11th week: 10.09.13 – 14.09.13====
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Cloning sfGFP N-terminal, GFP N-terminal and GFP C-terminal. Unfortunately fail. Checking buffers for digesting – they’re fine. Searching mistakes in cloning.
Cloning sfGFP N-terminal, GFP N-terminal and GFP C-terminal. Unfortunately fail. Checking buffers for digesting – they’re fine. Searching mistakes in cloning.
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====12th week: 16.09.13 – 20.09.13====
====12th week: 16.09.13 – 20.09.13====
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We get out commission from GeneRay. Cloning this to pSB1C3 plasmids, but it ends with failure. Cleaning lab and end work.
We get out commission from GeneRay. Cloning this to pSB1C3 plasmids, but it ends with failure. Cleaning lab and end work.
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{{:Team:Warsaw/Templates/StandardPageEnd}}
{{:Team:Warsaw/Templates/StandardPageEnd}}

Latest revision as of 18:22, 1 October 2013

Genetic lab journal

Contents


1st week: 2.07.13 – 5.07.13

Preparation of competent cells for chemical-based transfection. Failure.

2nd week: 8.07.13 – 12.07.13

Succeed preparation of competent cells for chemical-based transfection. Verification with iGEM’s transformation kit. Preparation of competent cells for electroporation. Failure.

3rd week: 15.07.13 – 19.07.13

Chemical-based transformation. BioBricks: J06504, J23100, B0034, I712074, E0040, B0015, B0017, E2020, E2030. Failure: E2020. Succeed: J06504, J23100, B0034, I712074, E0040, B0015, B0017, E2030. Isolate plasmid DNA by alkaline lysis. Quality cheked with spectrophotomeric analysis with NanoDrop.

4th week: 22.07.13 – 26.07.13

Transformation with isolated pladmids and BioBricks: I74609 and K864100. Isolate plasmid DNA (from I73609 and K864100) with mini-prep kit. Digest and ligation of construct: J23100 and B0034 on plasmid pSB1C3.

5th week: 29.07.13 – 1.08.13

Preparing construct: J23100 + B0034 + K864100 on plasmid pSB1A3. Measure glowing fluorescent proteins: sfGTP, sfYFP, sfCFP, sfBFP and SYFP2. Superfolded (sf) forms are made by Warsaw Team. SYFP2 is from parts registry.

6th week: 5.08.13 – 9.08.13=

Measure glowing fluorescent proteins. Banking strains with our fluorescent proteins.

7th week: 12.08.13 – 14.08.13

Transformation BioBrick: E2050. PCR: sfBFP N-terminal, sfYFP N-terminal and mCherry (J06504) C-terminal (succeed). Isolate plasmid DNA with: sfGFP (I73609), mOrange (E2050), sfCFP, sfYFP and sfBFP.

8th week: 19.08.13 – 23.08.13

PCR: sfGFP N-terminal (failure), sfCFP N-terminal (succeed) and mCherry N-terminal (succeed). Gel-out: sfYFP N-terminal, sfBFP N-terminal and mCherry C-terminal. Cloning sfCFP N-terminal, mCherry N-terminal, sfYFP N-terminal, sfBFP N-terminal and mCherry C-terminal to pSB1C3 plasmid and inserting it to bacteria. Preparing construct: J23100+B0034+E0040 – failure.

9th week: 26.08.13 – 30.08.13

Verification cloning

caption

Marker - GeneRuler 100 bp DNA Ladder, ready-to-use (Thermo); sfYFP N 1,2,3,R1,R2,R3 - random clones of sfYFP N-terminal; sfCFP N 1,2,3,R1,R2,R3 - random clones of sfCFP N-terminal; mCherry N 1,2,3,R1,R2,R3 - random clones of mCherry N-terminal; mCherry C 1,2,3,R1,R2,R3 - random clones of mCherry C-terminal; sfBFP N 1,2,3,R1,R2,R3 - random clones of sfBFP N-terminal. Underlined - correct clones.

And great success! PCR: sfGFP N-terminal and GFP C-terminal (various attenuation template DNA) and sfC-terminal (to versions: m6 and m12 to all of superfolded fluorescent proteins). Cloning and inserting it to bacteria. Once again preparing construct: J23100+B0034+E0040.

10th week: 2.09.13 – 7.09.13

Welcome school! Verification cloning. Unfortunately epic fail. PCR: sfGFP N-terminal, GFP N-terminal and GFP C-terminal. At least success!

11th week: 10.09.13 – 14.09.13

Cloning sfGFP N-terminal, GFP N-terminal and GFP C-terminal. Unfortunately fail. Checking buffers for digesting – they’re fine. Searching mistakes in cloning.

12th week: 16.09.13 – 20.09.13

We get out commission from GeneRay. Cloning this to pSB1C3 plasmids, but it ends with failure. Cleaning lab and end work.

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