Team:Warsaw/Journal

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{{:Team:Warsaw/Templates/StandardPageBegin|Genetic lab journal}}
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==Genetic lab diary==
 
====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.
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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====
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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.
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====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.
{{: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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