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Team:KAIST Korea/Notebook Labnote/2012 9
From 2012.igem.org
2012 KAIST Korea
Mail : kaist.igem.2012@gmail.com
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Notebook : Labnote-September
Labnote
SeptemberSeptember 1st 2012
Flip FlopBxb1_GTG_pBAD cloning with remaining DNAs
Remaining Bxb1_GTG insert was cloned into pBADmycHisC vector. And electrotransformed into MG1655.
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Back to the CalendarSeptember 2nd 2012
Flip FlopBxb1_ATG_pTrc, pBAD, Bxb1_GTG_pTrc miniprep for double transformation, Bxb1_GTG_pBAD colony inoculation and vector miniprep NcoI single cut check.
Results
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 FlopBxb1 double transformants vector miniprep & PCR check
Results
Discussions
Seemed to every colonies have appropriate vectors.
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September 4th 2012
No Special Event!
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September 5th 2012
No Special Event!
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September 6th 2012
No Special Event!
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September 7th 2012
No Special Event!
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September 8th 2012
No Special Event!
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September 9th 2012
No Special Event!
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September 10th 2012
Flip FlopBxb1_GTG_pBAD single cut check with different enzyme
Single cut with SnaBI
Results
No desired size(5.6kb) vector appeared.
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Flip Flop
Flip Flop
Flip Flop
Flip Flop
September 11th 2012
No Special Event!
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September 12th 2012
Flip FlopBxb1_GTG_pBAD colony #4~7 inoculation, miniprep and single cut check
Single cut with SnaBI
Single cut with SnaBI
Results
Cloning completed
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September 13th 2012
No Special Event!
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September 14th 2012
No Special Event!
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September 15th 2012
Flip FlopBBa_C0060, BBa_C0061, BBa_C0062 transformation into TOP10
Templates for Auto-regulated FlipFlop project.
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September 16th 2012
Flip FlopBBa_C0060, BBa_C0061, BBa_C0062, bFMO inoculation and enzyme cut check
BBa_C0060, BBa_C0061, BBa_C0062: from colony, EcoRI single cut
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- BBa_C0060: 2859bp
- BBa_C0061: 2688bp
- BBa_C0062: 2826bp
- bFMO: from cell stock, EcoRI single cut
September 17th 2012
Flip FlopTemplate preparation for OE PCR
Promoter construct: 210bp
Results
Promoter PCR failed
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Flip Flop
Flip Flop
Flip Flop
Flip Flop
Flip Flop
Flip Flop
Flip Flop
Flip Flop
September 18th 2012
Flip FlopTemplate preparation for OE PCR
Results
Terminator PCR failed
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September 19th 2012
Flip FlopTemplate preparation for OE PCR
Terminator: 179bp
Results
→ Gel extracted
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September 20th 2012
Flip FlopTemplate preparation for OE PCR
Bxb1 Xis: 777bp
Results
bFMO PCR w/ various condtions
Bxb1_Xis-pGEM_B1 transfomation
Synthesized gene – Bxb1_Xis arrived and transformed into TOP10
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September 21st 2012
No Special Event!
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September 22nd 2012
Flip FlopOverlapping extension PCR
Results
→ Gel extracted
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September 23rd 2012
Flip FlopOverlapping extension PCR-Nested PCR
Results
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
Discussions
Bands were more intense than those of first trial.
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September 24th 2012
Flip FlopOverlapping extension PCR Gel Extraction
Results
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P1, PI construct
Results
Full fragment for pAuto is overlapped. The right size(3794bp) fragments are gel extracted.
September 25th 2012
No Special Event!
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September 26th 2012
Flip FlopFull pAuto and pAutoSimple Overlapping PCR
Results
Discussion
The band intensity is too weak.
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pAutoIntegrase Overlapping PCR
Results
Full construct(2.5kb) for pAutoIntegrase is overlapped. The products are pcr purified and performed nested-pcr. 4th lane is the OE pcr product with rearranged template concentration.
Template preparation of terminator
Results
Terminator template for OE pcr is re-amplified with pfu-X DNA polymerase. The products are pcr purified.
September 27th 2012
No Special Event!
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September 28th 2012
No Special Event!
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September 29th 2012
Flip FlopTemplate preparation of bFMO (for pAuto and pAutoSimple)
Results
To find out the right temperature for primer binding, we performed gradient pcr from 45℃~55℃.
But, all the pcr products showed non-specific binding of primers, and we selected the most proper temperature.
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September 30th 2012
No Special Event!
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