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KAIST Korea 2012 iGEM

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2012 KAIST Korea

Mail : kaist.igem.2012@gmail.com
Twitter : twitter.com/KAIST_iGEM_2012
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Notebook : Labnote-September

Labnote

September

September 1^st 2012

　PACKMAN

fdh pBAD, fdh pTrc colony PCR check

Results

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 2^nd 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

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 3^rd 2012

　PACKMAN

1201 1197 1198 1516 PCR for Gibson assembly.

Results

Discussion

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

1516 PCR for Gibson assembly.

Results

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

Discussions

Seemed to every colonies have appropriate vectors.

Back to the Calendar

September 4^th 2012

　PACKMAN

1516 PCR for Gibson assembly.

Results

Discussion

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

Back to the Calendar

September 5^th 2012

　PACKMAN

1201 1197 1198 PCR amplification

Results

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

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. .

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September 6^th 2012

　PACKMAN

1198 1516PCR amplification

Results

Discussion

To amplify the amount of each fragments, we did PCR amplification of 1198 once more. And we did PCR amplification of 1516 gene once more with the same primers. For 1516 gene, we couldn’t have right size of band for new primers. So we decided to use the fragments of 1516 that does not contain terminator sequence.

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September 7^th 2012

No Special Event!

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September 8^th 2012

No Special Event!

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September 9^th 2012

No Special Event!

Back to the Calendar

September 10^th 2012

　Flip Flop

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

September 11^th 2012

No Special Event!

Back to the Calendar

September 12^th 2012

　Flip Flop

Bxb1_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 13^th 2012

No Special Event!

Back to the Calendar

September 14^th 2012

No Special Event!

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September 15^th 2012

　Flip Flop

BBa_C0060, BBa_C0061, BBa_C0062 transformation into TOP10

Templates for Auto-regulated FlipFlop project.

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September 16^th 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

Back to the Calendar

September 17^th 2012

　Flip Flop

Template preparation for OE PCR

Promoter construct: 210bp

Results

Promoter PCR failed

Back to the Calendar

September 18^th 2012

　Flip Flop

Template preparation for OE PCR

Results

Terminator PCR failed

Back to the Calendar

September 19^th 2012

　Flip Flop

Template preparation for OE PCR

Terminator: 179bp

Results

→ Gel extracted

Back to the Calendar

September 20^th 2012

　Flip Flop

Template 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 21^st 2012

No Special Event!

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September 22^nd 2012

　Flip Flop

Overlapping extension PCR

Results

→ Gel extracted

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September 23^rd 2012

　Flip Flop

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.

Overlapping extension PCR 2

Results

Discussions

Bands were more intense than those of first trial.

Back to the Calendar

September 24^th 2012

　Flip Flop

Overlapping extension PCR Gel Extraction

Results

P1, PI construct

Results

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

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September 25^th 2012

No Special Event!

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September 26^th 2012

　Flip Flop

Full pAuto and pAutoSimple Overlapping PCR

Results

Discussion

The band intensity is too weak.

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.

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September 27^th 2012

No Special Event!

Back to the Calendar

September 28^th 2012

No Special Event!

Back to the Calendar

September 29^th 2012

　Flip Flop

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.

Overlapping extension PCR Gel Extraction

Results

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September 30^th 2012

　Flip Flop

Template vector preparation – double digestion check with EcoRI and PstI

Results

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

bFMO template amplification with optimized condition

Results

pAutoIntegrase cloning check

Results

PCR amplified from prepped vector

Back to the Calendar

Kaist Footer

@@ Line 391: / Line 391: @@
 		<section id="6">
 			<div class="date">September 6<sup>th</sup> 2012</div></br>
+			<img style="height:3px;width:269px;margin:0px 5px 0px 0px;	"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:35px;float:bottom;"id="packman" src='https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png'/>　PACKMAN<img style="height:3px;width:262px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></br>
+			<div class="note-title"> <i>1198</i> <i>1516</i>PCR amplification</div>
 			<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="https://static.igem.org/mediawiki/2012/8/8c/120906.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 1198 once more. And we did PCR amplification of 1516 gene once more with the same primers. For 1516 gene, we couldn’t have right size of band for new primers. So we decided to use the fragments of 1516 that does not contain terminator sequence. </br></span>
 			</div>
 			<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>

Team:KAIST Korea/Notebook Labnote/2012 9

From 2012.igem.org

Revision as of 03:03, 27 October 2012

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