Team:KAIST Korea/Notebook Labnote/2012 9

From 2012.igem.org

(Difference between revisions)
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<section id="4">
<section id="4">
<div class="date">September 4<sup>th</sup> 2012</div></br>
<div class="date">September 4<sup>th</sup> 2012</div></br>
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<img style="height:3px;width:269px;margin:0px 5px 0px 0px; "src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:35px;float:bottom;"id="packman" src='http://2012.igem.org/wiki/images/0/03/KAIST_PACKMAN.png'/> PACKMAN<img style="height:3px;width:262px; margin:0px 0px 0px 5px;"src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/></br>
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<div class="note-title"> <i>1516</i> PCR for Gibson assembly.</div>
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<div id="content_note" >
<div id="content_note" >
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<span id="little">No Special Event!</span>
 
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</br>
 
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<b>Results</b></br></br>
 +
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<div align="center"><img id="figure" alt="0818Fig1" src="http://2012.igem.org/wiki/images/c/c1/120903-%2B--.PNG"></img></div>
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<div style="clear:both;"></div>
 +
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</br></br>
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<b>Discussion</b></br></br>
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<span id="little">Also for this time, there was no right size of band appeared for 1516 gene. </br></span>
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</div>
</div>
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
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<section id="5">
<section id="5">
<div class="date">September 5<sup>th</sup> 2012</div></br>
<div class="date">September 5<sup>th</sup> 2012</div></br>
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 +
<img style="height:3px;width:269px;margin:0px 5px 0px 0px; "src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:35px;float:bottom;"id="packman" src='http://2012.igem.org/wiki/images/0/03/KAIST_PACKMAN.png'/> PACKMAN<img style="height:3px;width:262px; margin:0px 0px 0px 5px;"src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/></br>
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<div class="note-title"> <i>1201</i> <i>1197</i> <i>1198</i> PCR amplification</div>
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<div id="content_note" >
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<b>Results</b></br></br>
 +
 +
<div align="center"><img id="figure" alt="0818Fig1" src="http://2012.igem.org/wiki/images/b/ba/120905_-_1.PNG"></img></div>
 +
<div style="clear:both;"></div>
 +
 +
</br></br>
 +
<b>Discussion</b></br></br>
 +
<span id="little">To amplify the amount of each fragment, we did PCR amplification of each gene once more. Correct size of bands appeared.  </br></span>
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 +
</div>
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</br><div align="center"><img style="width:670px; height:2px;" src="http://2012.igem.org/wiki/images/7/7f/Horizontal_line_kaist.png"></img></div></br>
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 +
<div class="note-title"><i>1197</i> <i>1516</i> PCR amplification </div>
 +
<div id="content_note" >
<div id="content_note" >
-
<span id="little">No Special Event!</span>
 
-
</br>
 
 +
<b>Results</b></br></br>
 +
 +
<div align="center"><img id="figure" alt="0818Fig1" src="http://2012.igem.org/wiki/images/9/94/120905-2.PNG"></img></div>
 +
<div style="clear:both;"></div>
 +
 +
</br></br>
 +
 +
<b>Discussion</b></br></br>
 +
 +
<span id="little">To amplify the amount of each fragments, we did PCR amplification of 1197 once more. And we did PCR amplification of 1516 gene once more with the same primers. Correct size of bands appeared for 1197 gene, but there was no band for 1516 gene. . </br></br></span>
 +
</div>
</div>
 +
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
</section>
</section>

Revision as of 03:01, 27 October 2012

KAIST Korea 2012 iGEM

Notebook : Labnote-September

Labnote

September

September 1st 2012

 PACKMAN
fdh pBAD, fdh pTrc colony PCR check
Results

0818Fig1


Discussion

To check the presence of FDH gene, we picked the colony from the plate and did colony PCR. For all the colonies we picked, we confirmed that correct size of FDH is present.
 Flip Flop

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

Back to the Calendar
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
Back to the Calendar
September 3rd 2012

 PACKMAN
1201 1197 1198 1516 PCR for Gibson assembly.
Results

0818Fig1


Discussion

There was no band for 1516 gene. Band of all three genes appeared at the right size.


1516 PCR for Gibson assembly.
Results

0818Fig1


Discussion

Also for this time, there was no right size of band appeared for 1516 gene.

We,
1. changed the fraction of primers down to 0.5ul each, (lane 1)
2. changed the polymerase to pfu-x, (lane3)
3. used hybrid primers ( TOPO forward, Gibson reverse : lane 4 and TOPO reverse, Gibson forward : lane 5)
Lane 2 was the control experiment with Hotstartaq polymerase.



Pre-culture of piBR181 vector containing cells.
Procedure

We did 3ml pre-culture on five 10ml tubes for the cells containing each gene. Cell was grown about 16hours after pre-culture, and then induced with proper inducer.

 Flip Flop

Bxb1 double transformants vector miniprep & PCR check
Results

2_0903Fig1

Discussions

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

 PACKMAN
1516 PCR for Gibson assembly.
Results

0818Fig1


Discussion

Also for this time, there was no right size of band appeared for 1516 gene.
Back to the Calendar
September 5th 2012

 PACKMAN
1201 1197 1198 PCR amplification
Results

0818Fig1


Discussion

To amplify the amount of each fragment, we did PCR amplification of each gene once more. Correct size of bands appeared.


1197 1516 PCR amplification
Results

0818Fig1


Discussion

To amplify the amount of each fragments, we did PCR amplification of 1197 once more. And we did PCR amplification of 1516 gene once more with the same primers. Correct size of bands appeared for 1197 gene, but there was no band for 1516 gene. .

Back to the Calendar
September 6th 2012

No Special Event!
Back to the Calendar
September 7th 2012

No Special Event!
Back to the Calendar
September 8th 2012

No Special Event!
Back to the Calendar
September 9th 2012

No Special Event!
Back to the Calendar
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.
Back to the Calendar
September 11th 2012

No Special Event!
Back to the Calendar
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
Back to the Calendar
September 13th 2012

No Special Event!
Back to the Calendar
September 14th 2012

No Special Event!
Back to the Calendar
September 15th 2012

 Flip Flop

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

Back to the Calendar
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

Back to the Calendar
September 17th 2012

 Flip Flop

Template preparation for OE PCR

Promoter construct: 210bp

Results

2_0917Fig1

Promoter PCR failed

2_0917Fig2
Back to the Calendar
September 18th 2012

 Flip Flop

Template preparation for OE PCR
Results

2_0918Fig1

Terminator PCR failed
Back to the Calendar
September 19th 2012

 Flip Flop

Template preparation for OE PCR
Terminator: 179bp

Results

2_0919Fig1

→ Gel extracted
Back to the Calendar
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

Back to the Calendar
September 21st 2012

No Special Event!
Back to the Calendar
September 22nd 2012

 Flip Flop

Overlapping extension PCR
Results

2_0922Fig1

→ Gel extracted
Back to the Calendar
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.
Back to the Calendar
September 24th 2012

 Flip Flop

Overlapping extension PCR Gel Extraction
Results

2_0924Fig1



P1, PI construct
Results

Full fragment for pAuto is overlapped. The right size(3794bp) fragments are gel extracted.

0818Fig1
Back to the Calendar
September 25th 2012

No Special Event!
Back to the Calendar
September 26th 2012

 Flip Flop

Full pAuto and pAutoSimple Overlapping PCR
Results

0818Fig1


Discussion

The band intensity is too weak.


pAutoIntegrase Overlapping PCR
Results

0818Fig1

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.

0818Fig1


Template preparation of terminator
Results

0818Fig1

Terminator template for OE pcr is re-amplified with pfu-X DNA polymerase. The products are pcr purified.
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September 27th 2012

No Special Event!
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September 28th 2012

No Special Event!
Back to the Calendar
September 29th 2012

 Flip Flop

Template preparation of bFMO (for pAuto and pAutoSimple)
Results

0818Fig1

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.


Overlapping extension PCR Gel Extraction
Results

0818Fig1


Back to the Calendar
September 30th 2012

 Flip Flop

Template vector preparation – double digestion check with EcoRI and PstI
Results

0818Fig1

Vector(BBa_J04450-pSB4A5) which will be used for cloning of pAutoIntegrase.


bFMO template amplification with optimized condition
Results

0818Fig1


pAutoIntegrase cloning check
Results

PCR amplified from prepped vector
0818Fig1
Back to the Calendar


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