Team:KAIST Korea/Notebook Labnote/2012 9

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KAIST Korea 2012 iGEM

Notebook : Labnote-September

Labnote

September

September 1st 2012

 Flip Flop

Bxb1_GTG_pBAD cloning with remaining DNAs
Remaining Bxb1_GTG insert was cloned into pBADmycHisC vector. And electrotransformed into MG1655.

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September 2nd 2012

 Flip Flop

Bxb1_ATG_pTrc, pBAD, Bxb1_GTG_pTrc miniprep for double transformation, Bxb1_GTG_pBAD colony inoculation and vector miniprep NcoI single cut check.
Results

2_0902Fig1

Discussions

All the vectors showed right sizes. But Bxb1_GTG_pBAD colony #1 through 3 have undesired bands. They may be the supercoiled vector.


Bxb1_ATG_pTrc, pBAD, Bxb1_GTG_pTrc double transformation into pPoC and pPoCpi
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September 3rd 2012

 Flip Flop

Bxb1 double transformants vector miniprep & PCR check
Results

2_0903Fig1

Discussions

Seemed to every colonies have appropriate vectors.
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September 4th 2012

September 5th 2012

September 6th 2012

September 7th 2012

September 8th 2012

September 9th 2012

September 10th 2012

 Flip Flop

Bxb1_GTG_pBAD single cut check with different enzyme
Single cut with SnaBI

Results

2_0910Fig1

No desired size(5.6kb) vector appeared.
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September 11th 2012

September 12th 2012

 Flip Flop

Bxb1_GTG_pBAD colony #4~7 inoculation, miniprep and single cut check Single cut with SnaBI
Single cut with SnaBI

Results

2_0912Fig1

Cloning completed
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September 13th 2012

September 14th 2012

September 15th 2012

 Flip Flop

BBa_C0060, BBa_C0061, BBa_C0062 transformation into TOP10
Templates for Auto-regulated FlipFlop project.

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September 16th 2012

 Flip Flop

BBa_C0060, BBa_C0061, BBa_C0062, bFMO inoculation and enzyme cut check
BBa_C0060, BBa_C0061, BBa_C0062: from colony, EcoRI single cut

  • BBa_C0060: 2859bp
  • BBa_C0061: 2688bp
  • BBa_C0062: 2826bp
  • bFMO: from cell stock, EcoRI single cut


Results

2_0916#Fig#1

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September 17th 2012

 Flip Flop

Template preparation for OE PCR

Promoter construct: 210bp
Results

2_0917Fig1
Promoter PCR failed
2_0917Fig2
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September 18th 2012

 Flip Flop

Template preparation for OE PCR
Results

2_0918Fig1

Terminator PCR failed
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September 19th 2012

 Flip Flop

Template preparation for OE PCR
Terminator: 179bp

Results

2_0919Fig1

→ Gel extracted
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September 20th 2012

 Flip Flop

Template preparation for OE PCR
Bxb1 Xis: 777bp

Results

2_0920Fig1
bFMO PCR w/ various condtions
2_0920Fig2



Bxb1_Xis-pGEM_B1 transfomation
Synthesized gene – Bxb1_Xis arrived and transformed into TOP10

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September 21st 2012

September 22nd 2012

 Flip Flop

Overlapping extension PCR
Results

2_0922Fig1

→ Gel extracted
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September 23rd 2012

 Flip Flop

Overlapping extension PCR-Nested PCR
Results

2_0923Fig1

Discussions

There was too little full construct nested. We think this is due to primer interfering by its LVA homology.


Overlapping extension PCR 2
Results

2_0923Fig2

Discussions

Bands were more intense than those of first trial.
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September 24th 2012

 Flip Flop

Overlapping extension PCR Gel Extraction
Results

2_0924Fig1



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September 25th 2012

September 26th 2012

September 27th 2012

September 28th 2012

September 29th 2012

September 30th 2012

September 31st 2012



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