Team:KAIST Korea/Notebook Labnote/2012 9
From 2012.igem.org
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<span id="little"></br>Vector(BBa_J04450-pSB4A5) which will be used for cloning of pAutoIntegrase.</br></span> | <span id="little"></br>Vector(BBa_J04450-pSB4A5) which will be used for cloning of pAutoIntegrase.</br></span> | ||
+ | |||
+ | </br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br> | ||
+ | <div class="note-title">bFMO template amplification with optimized condition</div> | ||
+ | |||
+ | <b>Results</b></br></br> | ||
+ | <div id="content_note" > | ||
+ | <div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/e/e1/120930-.1.PNG"></img></div> | ||
+ | <div style="clear:both;"></div> | ||
+ | |||
+ | </div> | ||
+ | |||
+ | </br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br> | ||
+ | <div class="note-title">pAutoIntegrase cloning check</div> | ||
+ | <b>Results</b></br></br> | ||
+ | <span id="little">PCR amplified from prepped vector </br></br> | ||
+ | </span> | ||
+ | |||
+ | <div id="content_note" > | ||
+ | <div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/2/27/120930%3D2.PNG"></img></div> | ||
+ | <div style="clear:both;"></div> | ||
+ | |||
+ | </div> | ||
+ | |||
</div> | </div> | ||
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Revision as of 00:49, 27 October 2012
![](https://static.igem.org/mediawiki/2012/5/59/Logopart_Kaist.png)
![](https://static.igem.org/mediawiki/2012/6/61/Home_kaist_background.png)
2012 KAIST Korea
Mail : kaist.igem.2012@gmail.com
Twitter : twitter.com/KAIST_iGEM_2012
Facebook : www.facebook.com/KAISTiGEM2012
Notebook : Labnote-September
![](https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png)
![](https://static.igem.org/mediawiki/2012/9/95/Starter_gradient_kaist.png)
Labnote
SeptemberSeptember 1st 2012
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png)
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Bxb1_GTG_pBAD cloning with remaining DNAs
Remaining Bxb1_GTG insert was cloned into pBADmycHisC vector. And electrotransformed into MG1655.
Back to the CalendarSeptember 2nd 2012
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png)
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Bxb1_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.
![2_0902Fig1](https://static.igem.org/mediawiki/2012/1/1c/KAIST_2_Sept.png)
![](https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png)
Bxb1_ATG_pTrc, pBAD, Bxb1_GTG_pTrc double transformation into pPoC and pPoCpi
Back to the CalendarSeptember 3rd 2012
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png)
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Bxb1 double transformants vector miniprep & PCR check
Results
Discussions
Seemed to every colonies have appropriate vectors.
Back to the CalendarSeptember 4th 2012
No Special Event!
Back to the CalendarSeptember 5th 2012
No Special Event!
Back to the CalendarSeptember 6th 2012
No Special Event!
Back to the CalendarSeptember 7th 2012
No Special Event!
Back to the CalendarSeptember 8th 2012
No Special Event!
Back to the CalendarSeptember 9th 2012
No Special Event!
Back to the CalendarSeptember 10th 2012
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png)
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Bxb1_GTG_pBAD single cut check with different enzyme
Single cut with SnaBI
Results
No desired size(5.6kb) vector appeared.
Back to the Calendar
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Flip Flop
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Flip Flop
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Flip Flop
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Flip Flop
September 11th 2012
No Special Event!
Back to the CalendarSeptember 12th 2012
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png)
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Bxb1_GTG_pBAD colony #4~7 inoculation, miniprep and single cut check
Single cut with SnaBI
Single cut with SnaBI
Results
Cloning completed
Back to the Calendar![2_0912Fig1](https://static.igem.org/mediawiki/2012/1/13/KAIST_12_Sept.png)
September 13th 2012
No Special Event!
Back to the CalendarSeptember 14th 2012
No Special Event!
Back to the CalendarSeptember 15th 2012
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png)
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
BBa_C0060, BBa_C0061, BBa_C0062 transformation into TOP10
Templates for Auto-regulated FlipFlop project.
Back to the CalendarSeptember 16th 2012
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png)
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
BBa_C0060, BBa_C0061, BBa_C0062, bFMO inoculation and enzyme cut check
BBa_C0060, BBa_C0061, BBa_C0062: from colony, EcoRI single cut
Back to the Calendar- BBa_C0060: 2859bp
- BBa_C0061: 2688bp
- BBa_C0062: 2826bp
- bFMO: from cell stock, EcoRI single cut
September 17th 2012
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png)
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Template preparation for OE PCR
Promoter construct: 210bp
Results
Promoter PCR failed
Back to the Calendar
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Flip Flop
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Flip Flop
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Flip Flop
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Flip Flop
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Flip Flop
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Flip Flop
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Flip Flop
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Flip Flop
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Flip Flop
September 18th 2012
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png)
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Template preparation for OE PCR
Results
Terminator PCR failed
Back to the CalendarSeptember 19th 2012
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png)
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Template preparation for OE PCR
Terminator: 179bp
Results
→ Gel extracted
Back to the CalendarSeptember 20th 2012
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png)
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Template preparation for OE PCR
Bxb1 Xis: 777bp
Results
bFMO PCR w/ various condtions
![](https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png)
Bxb1_Xis-pGEM_B1 transfomation
Synthesized gene – Bxb1_Xis arrived and transformed into TOP10
Back to the CalendarSeptember 21st 2012
No Special Event!
Back to the CalendarSeptember 22nd 2012
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png)
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Overlapping extension PCR
Results
→ Gel extracted
Back to the CalendarSeptember 23rd 2012
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png)
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Overlapping 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.
![](https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png)
Overlapping extension PCR 2
Results
Discussions
Bands were more intense than those of first trial.
Back to the CalendarSeptember 24th 2012
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png)
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Overlapping extension PCR Gel Extraction
Results
Back to the Calendar![](https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png)
P1, PI construct
Results
Full fragment for pAuto is overlapped. The right size(3794bp) fragments are gel extracted.
September 25th 2012
No Special Event!
Back to the CalendarSeptember 26th 2012
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png)
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Full pAuto and pAutoSimple Overlapping PCR
Results
Discussion
The band intensity is too weak.
Back to the Calendar![](https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png)
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.
![](https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png)
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!
Back to the CalendarSeptember 28th 2012
No Special Event!
Back to the CalendarSeptember 29th 2012
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png)
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Template 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.
Back to the Calendar![](https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png)
Overlapping extension PCR Gel Extraction
Results
September 30th 2012
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png)
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Template vector preparation – double digestion check with EcoRI and PstI
Results
Vector(BBa_J04450-pSB4A5) which will be used for cloning of pAutoIntegrase.
Back to the Calendar![](https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png)
bFMO template amplification with optimized condition
Results
![](https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png)
pAutoIntegrase cloning check
Results
PCR amplified from prepped vector