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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
Facebook : www.facebook.com/KAISTiGEM2012

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

Back to the Calendar

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. .

Back to the Calendar

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.

Back to the Calendar

September 7^th 2012

　PACKMAN

Pre-culture of 0109, 1191, FDH containing cells.

Discussion

We did 5ml pre-culture on 10ml tube for the cells containing each gene. Cell was grown about 16hours after pre-culture, and then induced with proper inducer.

Back to the Calendar

September 8^th 2012

　PACKMAN

FDH pTrc & pBAD/0109 pBAD/1191 pBAD MG1655 expression check (sampling and induction)

Procedure

We seeded cells (500λ of cells put into 50mL LB with 50λ Ampicillin) and checked Optical Density. According to the O.D. value, we induced 37℃ and 30℃ sample and did sampling. Each samples were induced by different conditions ; 1mM and 2mM of IPTG, 10mM and 30mM of arabinose.

Back to the Calendar

September 9^th 2012

　PACKMAN

Moth_0109 expression optimization _ gel picture

Results

Discussion

For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 59.94kDa protein, formate--tetrahydrofolate ligase. The gel was ruined during staining/destaining procedure, and we couldn’t find any band for this protein.
Therefore, we concluded that expression of 0109 gene on pBAD vector has failed and decided to try once more.

Moth_1191 expression optimization _ gel picture

Results

Discussion

For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 33.92kDa protein, methylenetetrahydrofolate reductase. The gel was ruined during staining/destaining procedure, and we couldn’t find any band for this protein.
Therefore, we concluded that expression of 1191 gene on pBAD vector has failed and decided to try once more.

FDH pTrc expression optimization _ gel picture

Results

Discussion

For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 40.37kDa protein, Formate dehydrogenase. The gel was ruined during staining/destaining procedure, and we couldn’t find any band for this protein.
Therefore, we concluded that expression of FDH gene on pTrc vector has failed and decided to try once more.

FDH pBAD expression optimization _ gel picture

Results

Discussion

For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 40.37kDa protein, Formate dehydrogenase. The gel was ruined during staining/destaining procedure, and we couldn’t find any band for this protein.
Therefore, we concluded that expression of FDH gene on pTrc vector has failed and decided to try once more.

Primer design to PCR-amplify piBR181 vector.

Results

Discussion

For the Gibson assembly with RE digestion of vector, we had several problems with de-phosphorylation and purification after de-phosphorylation. Therefore we decided to PCR-amplify the piBR181 vector and use the PCR product in Gibson assembly.

Back to the Calendar

September 10^th 2012

　PACKMAN

PCR amplification of 1202 gene with new primers

Results

Discussion

The correct size of band appeared for the second PCR product, but there was no band appeared for the first PCR product.

　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

　PACKMAN

PCR amplification of piBR181 vector

Results

Discussion

The correct size of piBR181 vector pcr product is 5297bp. There appeared the right size and unspecific band on the gel. So we decided to do pcr once more.

Pre-culture of 0109, 1191, FDH containing cells.

Procedure

We did 5ml pre-culture on 10ml tube for the cells containing each gene. Cell was grown about 16hours after pre-culture, and then induced with proper inducer.

Back to the Calendar

September 12^th 2012

　PACKMAN

PCR amplification of piBR181 vector : 10 sample

Results

Discussion

The full PCR construct of piBR181 vector is 5297bp. From the gel image, we could find that right size of piBR181 PCR product clearly exists.

FDH pTrc & pBAD/0109 pBAD/1191 pBAD MG1655 expression check (sampling and induction)

Procedure

We seeded cells (500λ of cells put into 50mL LB with 50λ Ampicillin) and checked Optical Density. According to the O.D. value, we induced 37℃ and 30℃ sample and did sampling. Each samples were induced by different conditions ; 1mM and 2mM of IPTG, 10mM and 30mM of arabinose.

　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

Back to the Calendar

September 13^th 2012

　PACKMAN

Moth_0109 pBAD expression check

Results

Discussion

For each samples of different induction conditions, we ran 10% SDS-PAGE gel and checked the presence of 59.94kDa protein, formate--tetrahydrofolate ligase. We couldn’t find any band for this protein.
There was no significant band on the gel, so we couldn’t conclude that this protein is expressed. Our opinion is that we should run Western blot to exactly find this protein.

Moth_1191 pBAD expression check

Results

Discussion

For each samples of different induction conditions, we run 15% SDS-PAGE gel and checked the presence of 33.92kDa protein, methylenetetrahydrofolate reductase. Although we were not able to discriminate each band of insoluble fraction, We could find that there exists right band at the right size.

FDH pBAD expression check

Results

Discussion

For each samples of different induction conditions, we run 8% SDS-PAGE gel and checked the presence of 40.37 kDa protein, Formate dehydrogenase. We couldn’t find any band for this protein.
There was no significant band on the gel, so we couldn’t conclude that this protein is expressed. Our opinion is that we should run Western blot to exactly find this protein.

FDH pTrc expression check

Results

Discussion

For each samples of different induction conditions, we run 8% SDS-PAGE gel and checked the presence of 40.37 kDa protein, Formate dehydrogenase. We couldn’t find any band for this protein.
There was no significant band on the gel, so we couldn’t conclude that this protein is expressed. Our opinion is that we should run Western blot to exactly find this protein.

Back to the Calendar

September 14^th 2012

　PACKMAN

Gibson assembly – assembly of pcr fragments (pcr fragment of vector added)

Results

Among three methods to assemble DNA fragments, we chose third method, which is assembly of PCR amplified DNA fragments and vector. 0.17umoles of each fragments are added into the reaction sample, and volume to be added for each fragments has calculated on the assumption that average MW of each base is 660Da. Table below is the result of calculation.

After 1 hour of incubation on 50℃, the vector was transformed into TOP10 and NEB competent cell. Cells were plated and grown overnight during colony formation.

Back to the Calendar

September 15^th 2012

　Flip Flop

BBa_C0060, BBa_C0061, BBa_C0062 transformation into TOP10

Templates for Auto-regulated FlipFlop project.

Back to the Calendar

September 16^th 2012

　PACKMAN

Gibson assembly colony pcr

Results

Discussion

As we couldn’t get full fragment by colony-pcr, we did 3ml culture, plasmid mini-prep and PCR for each colony. This will take one more day to get result.

　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

　PACKMAN

Gibson assembly fragment PCR after prep

Results

Discussion

There appeared correct size of band with several unspecific bands on the gel. We couldn’t even try to do gel extraction because the band was too thin and faint. We decided to do more rounds of pcr amplification.

　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

　PACKMAN

Gibson F1 PCR after prep

Results

Discussion

We did PCR amplification after prep of F1 vector, but there appeared no band on the gel.

Gibson F2 5-1~5, F1-3-1~5, F1-4-1~5

Results

Discussion

There appeared several unspecific bands on the gel and

　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

Back to the Calendar

September 21^st 2012

No Special Event!

Back to the Calendar

September 22^nd 2012

　Flip Flop

Overlapping extension PCR

Results

→ Gel extracted

Back to the Calendar

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.

Back to the Calendar

September 25^th 2012

No Special Event!

Back to the Calendar

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.

Back to the Calendar

September 27^th 2012

　PACKMAN

Pre-culture of Gibson F1, F2 colonies

Results

We checked that there were several colonies on the plate. 10 colonies for each fragment were picked, and culture into 3mL LB media containing 30ul Kanamycin.

Back to the Calendar

September 28^th 2012

　PACKMAN

Vector preparation and PCR

Results

For each cultured cells, vectors were mini-prepped, and pcr was done with proper primers (F1 : 1201 F, 1197R, F2 : 1198F, 1516R)

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

Back to the Calendar

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

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 		<section id="1">
 			<div class="date">September 1<sup>st</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"> fdh pBAD, fdh pTrc colony PCR check</div>
+			<div id="content_note" >
+				<b>Results</b></br></br>
+				<div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/5/55/120901.PNG"></img></div>
+				<div style="clear:both;"></div>
+				</br></br>
+				<b>Discussion</b></br></br>
+				<span id="little">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. </br></span>
+			</div>
 			<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><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:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/>　Flip Flop<img style="height:3px;width:269px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
@@ Line 233: / Line 251: @@
 		<section id="3">
 			<div class="date">September 3<sup>rd</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>1201</i> <i>1197</i> <i>1198</i> <i>1516</i> PCR for Gibson assembly.</div>
+			<div id="content_note" >
+				<b>Results</b></br></br>
+				<div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/5/5e/120903.PNG"></img></div>
+				<div style="clear:both;"></div>
+				</br></br>
+				<b>Discussion</b></br></br>
+				<span id="little">There was no band for 1516 gene. Band of all three genes appeared at the right size.</br></span>
+			</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"><i>1516</i> PCR for Gibson assembly.</div>
+			<div id="content_note" >
+				<b>Results</b></br></br>
+				<div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/c/c1/120903-%2B--.PNG"></img></div>
+				<div style="clear:both;"></div>
+				</br></br>
+				<b>Discussion</b></br></br>
+				<span id="little">Also for this time, there was no right size of band appeared for 1516 gene. </br></br>
+We, </br>
+. changed the fraction of primers down to 0.5ul each, (lane 1)</br>
+. changed the polymerase to pfu-x, (lane3)</br>
+. used hybrid primers ( TOPO forward, Gibson reverse : lane 4 and TOPO reverse, Gibson forward : lane 5)</br>
+Lane 2 was the control experiment with Hotstartaq polymerase.</br>
+</br></span>
+			</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">Pre-culture of piBR181 vector containing cells. </div>
+			<div id="content_note" >
+				<b>Procedure</b></br></br>
+				<span id="little">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. </br></br></span>
+			</div>
 				<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><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:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/>　Flip Flop<img style="height:3px;width:269px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
@@ Line 261: / Line 332: @@
 		<section id="4">
 			<div class="date">September 4<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>1516</i> PCR for Gibson assembly.</div>
+			<div id="content_note" >
+				<b>Results</b></br></br>
+				<div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/c/c1/120903-%2B--.PNG"></img></div>
+				<div style="clear:both;"></div>
+				</br></br>
+				<b>Discussion</b></br></br>
+				<span id="little">Also for this time, there was no right size of band appeared for 1516 gene. </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>
 		</section>
 		<section id="5">
 			<div class="date">September 5<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>1201</i> <i>1197</i> <i>1198</i> PCR amplification</div>
+			<div id="content_note" >
+				<b>Results</b></br></br>
+				<div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/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>
+			</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"><i>1197</i> <i>1516</i> PCR amplification </div>
+			<div id="content_note" >
+				<b>Results</b></br></br>
+				<div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/9/94/120905-2.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>
+			<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>
 		</section>
 		<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" >
+				<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>
 		</section>
 		<section id="7">
 			<div class="date">September 7<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">Pre-culture of <i>0109</i>, <i>1191</i>, FDH containing cells.</div>
+			<div id="content_note" >
+				<b>Discussion</b></br></br>
+				<span id="little">We did 5ml pre-culture on 10ml tube for the cells containing each gene. Cell was grown about 16hours after pre-culture, and then induced with proper inducer.
+ </br></br></span>
+			<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>
 		</section>
 		<section id="8">
 			<div class="date">September 8<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">FDH pTrc & pBAD/<i>0109</i> pBAD/<i>1191</i> pBAD MG1655 expression check (sampling and induction)</div>
+			<div id="content_note" >
+				<b>Procedure</b></br></br>
+				<span id="little">We seeded cells (500λ of cells put into 50mL LB with 50λ Ampicillin) and checked Optical Density. According to the O.D. value, we induced 37℃ and 30℃ sample  and did sampling. Each samples were induced by different conditions ; 1mM and 2mM of IPTG, 10mM and 30mM of arabinose.</br></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>
 		</section>
 		<section id="9">
 			<div class="date">September 9<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">Moth_0109 expression optimization _ gel picture</div>
+			<div id="content_note" >
+				<b>Results</b></br></br>
+				<div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/6/64/120909.PNG"></img></div>
+				<div style="clear:both;"></div>
+				<b>Discussion</b></br></br>
+				<span id="little">For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 59.94kDa protein, formate--tetrahydrofolate ligase. The gel was ruined during staining/destaining procedure, and we couldn’t find any band for this protein. </br>
+Therefore, we concluded that expression of 0109 gene on pBAD vector has failed and decided to try once more.</br></br></span>
+</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">Moth_1191 expression optimization _ gel picture </div>
+			<div id="content_note" >
+				<b>Results</b></br></br>
+				<div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/e/e0/120909_-_1.PNG"></img></div>
+				<div style="clear:both;"></div>
+				</br></br>
+				<b>Discussion</b></br></br>
+				<span id="little">For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 33.92kDa protein, methylenetetrahydrofolate reductase. The gel was ruined during staining/destaining procedure, and we couldn’t find any band for this protein. </br>
+Therefore, we concluded that expression of 1191 gene on pBAD vector has failed and decided to try once more.
+ </br></br></span>
+			</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">FDH pTrc expression optimization _ gel picture </div>
+			<div id="content_note" >
+				<b>Results</b></br></br>
+				<div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/b/bf/120909-2.PNG"></img></div>
+				<div style="clear:both;"></div>
+				</br></br>
+				<b>Discussion</b></br></br>
+				<span id="little">For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 40.37kDa protein, Formate dehydrogenase. The gel was ruined during staining/destaining procedure, and we couldn’t find any band for this protein. </br>
+Therefore, we concluded that expression of FDH gene on pTrc vector has failed and decided to try once more.
+ </br></br></span>
+			</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">FDH pBAD expression optimization _ gel picture</div>
+			<div id="content_note" >
+				<b>Results</b></br></br>
+				<div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/6/6d/120909-3.PNG"></img></div>
+				<div style="clear:both;"></div>
+				</br></br>
+				<b>Discussion</b></br></br>
+				<span id="little">For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 40.37kDa protein, Formate dehydrogenase. The gel was ruined during staining/destaining procedure, and we couldn’t find any band for this protein. </br>
+Therefore, we concluded that expression of FDH gene on pTrc vector has failed and decided to try once more.
+ </br></br></span>
+			</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">Primer design to PCR-amplify piBR181 vector.</div>
+			<div id="content_note" >
+				<b>Results</b></br></br>
+				<div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/d/d5/1209094.PNG"></img></div>
+				<div style="clear:both;"></div>
+				</br></br>
+				<b>Discussion</b></br></br>
+				<span id="little">For the Gibson assembly with RE digestion of vector, we had several problems with de-phosphorylation and purification after de-phosphorylation. Therefore we decided to PCR-amplify the piBR181 vector and use the PCR product in Gibson assembly. </br></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>
 		</section>
 		<section id="10">
 			<div class="date">September 10<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">PCR amplification of 1202 gene with new primers</div>
+			<div id="content_note" >
+				<b>Results</b></br></br>
+				<div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/a/ad/120910.PNG"></img></div>
+				<div style="clear:both;"></div>
+				<b>Discussion</b></br></br>
+				<span id="little">The correct size of band appeared for the second PCR product, but there was no band appeared for the first PCR product.  </br></br></span>
+			</div>
 			<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><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:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/>　Flip Flop<img style="height:3px;width:269px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
@@ Line 309: / Line 584: @@
 		<section id="11">
 			<div class="date">September 11<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">PCR amplification of piBR181 vector</div>
+			<div id="content_note" >
+				<b>Results</b></br></br>
+				<div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/6/66/120911.PNG"></img></div>
+				<div style="clear:both;"></div>
+				<b>Discussion</b></br></br>
+				<span id="little">The correct size of piBR181 vector pcr product is 5297bp. There appeared the right size and unspecific band on the gel. So we decided to do pcr once more.</br></br></span>
+			</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">Pre-culture of 0109, 1191, FDH containing cells.</div>
+			<div id="content_note" >
+				<b>Procedure</b></br></br>
+				<span id="little">We did 5ml pre-culture on 10ml tube for the cells containing each gene. Cell was grown about 16hours after pre-culture, and then induced with proper inducer.</br></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>
 		</section>
 		<section id="12">
-			<div class="date">September 12<sup>th</sup> 2012</div></br>
+		<div class="date">September 12<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">PCR amplification of piBR181 vector : 10 sample</div>
+			<div id="content_note" >
+				<b>Results</b></br></br>
+				<div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/6/65/120912.PNG"></img></div>
+				<div style="clear:both;"></div>
+				<b>Discussion</b></br></br>
+				<span id="little">The full PCR construct of piBR181 vector is 5297bp. From the gel image, we could find that right size of piBR181 PCR product clearly exists. </br></br></span>
+			</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">FDH pTrc & pBAD/0109 pBAD/1191 pBAD MG1655 expression check (sampling and induction)</div>
+			<div id="content_note" >
+				<b>Procedure</b></br></br>
+				<span id="little">We seeded cells (500λ of cells put into 50mL LB with 50λ Ampicillin) and checked Optical Density. According to the O.D. value, we induced 37℃ and 30℃ sample  and did sampling. Each samples were induced by different conditions ; 1mM and 2mM of IPTG, 10mM and 30mM of arabinose.</br></br></span>
+			</div>
 <div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><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:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/>　Flip Flop<img style="height:3px;width:269px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
@@ Line 341: / Line 674: @@
 		<section id="13">
 			<div class="date">September 13<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">Moth_0109 pBAD expression check</div>
+			<div id="content_note" >
+		<b>Results</b></br></br>
+				<div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/d/da/120913_-_1.PNG"></img></div>
+				<div style="clear:both;"></div>
+				<b>Discussion</b></br></br>
+				<span id="little">For each samples of different induction conditions, we ran 10% SDS-PAGE gel and checked the presence of 59.94kDa protein, formate--tetrahydrofolate ligase. We couldn’t find any band for this protein. </br>
+There was no significant band on the gel, so we couldn’t conclude that this protein is expressed. Our opinion is that we should run Western blot to exactly find this protein. </br></br></span>
+</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">Moth_1191 pBAD expression check</div>
+			<div id="content_note" >
+				<b>Results</b></br></br>
+				<div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/a/af/120913-2.PNG"></img></div>
+				<div style="clear:both;"></div>
+				</br></br>
+				<b>Discussion</b></br></br>
+				<span id="little">For each samples of different induction conditions, we run 15% SDS-PAGE gel and checked the presence of 33.92kDa protein, methylenetetrahydrofolate reductase. Although we were not able to discriminate each band of insoluble fraction, We could find that there exists right band at the right size.
+ </br></br></span>
+			</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">FDH pBAD expression check</div>
+			<div id="content_note" >
+				<b>Results</b></br></br>
+				<div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/9/9e/120913-3.PNG"></img></div>
+				<div style="clear:both;"></div>
+				</br></br>
+				<b>Discussion</b></br></br>
+				<span id="little">For each samples of different induction conditions, we run 8% SDS-PAGE gel and checked the presence of 40.37 kDa protein, Formate dehydrogenase. We couldn’t find any band for this protein.  </br>
+There was no significant band on the gel, so we couldn’t conclude that this protein is expressed. Our opinion is that we should run Western blot to exactly find this protein.
+ </br></br></span>
+			</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">FDH pTrc expression check</div>
+			<div id="content_note" >
+				<b>Results</b></br></br>
+				<div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/1/1b/120913-4.PNG"></img></div>
+				<div style="clear:both;"></div>
+				</br></br>
+				<b>Discussion</b></br></br>
+				<span id="little">For each samples of different induction conditions, we run 8% SDS-PAGE gel and checked the presence of 40.37 kDa protein, Formate dehydrogenase. We couldn’t find any band for this protein. </br>
+There was no significant band on the gel, so we couldn’t conclude that this protein is expressed. Our opinion is that we should run Western blot to exactly find this protein.
+ </br></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>
 		</section>
 		<section id="14">
 			<div class="date">September 14<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">Gibson assembly – assembly of pcr fragments (pcr fragment of vector added)</div>
+			<div id="content_note" >
+		<b>Results</b></br></br>
+				<span id="little">Among three methods to assemble DNA fragments, we chose third method, which is assembly of PCR amplified DNA fragments and vector.
+.17umoles of each fragments are added into the reaction sample, and volume to be added for each fragments has calculated on the assumption that average MW of each base is 660Da. Table below is the result of calculation.</br></br></span>
+				<div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/b/b7/120914_f1.PNG"></img></div>
+				<div style="clear:both;"></div>
+				<div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/8/8b/120914_f2.PNG"></img></div>
+				<div style="clear:both;"></div>
+				<span id="little"></br>After 1 hour of incubation on 50℃, the vector was transformed into TOP10 and NEB competent cell. Cells were plated and grown overnight during colony formation. </br></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>
 		</section>
 		<section id="15">
@@ Line 365: / Line 794: @@
 		<section id="16">
 			<div class="date">September 16<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">Gibson assembly colony pcr</div>
+			<div id="content_note" >
+		<b>Results</b></br></br>
+				<div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/f/fb/120916.PNG"></img></div>
+				<div style="clear:both;"></div>
+				<b>Discussion</b></br></br>
+				<span id="little">As we couldn’t get full fragment by colony-pcr, we did 3ml culture, plasmid mini-prep and PCR for each colony. This will take one more day to get result.</br></br></span>
+</div>
 						<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><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:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/>　Flip Flop<img style="height:3px;width:269px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
@@ Line 397: / Line 840: @@
 		<section id="17">
 			<div class="date">September 17<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">Gibson assembly fragment PCR after prep</div>
+			<div id="content_note" >
+		<b>Results</b></br></br>
+				<div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/f/f2/120917.PNG"></img></div>
+				<div style="clear:both;"></div>
+				<b>Discussion</b></br></br>
+				<span id="little">There appeared correct size of band with several unspecific bands on the gel. We couldn’t even try to do gel extraction because the band was too thin and faint. We decided to do more rounds of pcr amplification.</br></br></span>
+</div>
 			<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><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:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/>　Flip Flop<img style="height:3px;width:269px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
@@ Line 405: / Line 862: @@
 			</br>
 				<span id="little">Promoter construct: 210bp</span>
-				</br>
+				</br></br>
 				<b>Results</b></br></br>
 				<div align="center"><img id="figure" alt="2_0917Fig1" src="https://static.igem.org/mediawiki/2012/d/d3/KAIST_17_Sept.JPG"></img></div>
-				<div style="clear:both;"></div>
+				<div style="clear:both;"></div></br>
 				<span id="little">Promoter PCR failed</span>
-				</br>
+				</br></br>
 				<div align="center"><img id="figure" alt="2_0917Fig2" src="https://static.igem.org/mediawiki/2012/d/d7/KAIST_17_Sept_2.JPG"></img>
@@ Line 452: / Line 909: @@
 		<section id="19">
 			<div class="date">September 19<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">Gibson F1 PCR after prep</div>
+			<div id="content_note" >
+		<b>Results</b></br></br>
+				<div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/e/eb/120919-1.PNG"></img></div>
+				<div style="clear:both;"></div>
+				<b>Discussion</b></br></br>
+				<span id="little">We did PCR amplification after prep of F1 vector, but there appeared no band on the gel. </br></br></span>
+</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">Gibson F2 5-1~5, F1-3-1~5, F1-4-1~5</div>
+			<div id="content_note" >
+		<b>Results</b></br></br>
+				<div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/3/3c/120919-2.PNG"></img></div>
+				<div style="clear:both;"></div>
+				<b>Discussion</b></br></br>
+				<span id="little">There appeared several unspecific bands on the gel and  </br></br></span>
+</div>
 				<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><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:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/>　Flip Flop<img style="height:3px;width:269px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
@@ Line 492: / Line 977: @@
 				<div align="center"><img id="figure" alt="2_0920Fig1" src="https://static.igem.org/mediawiki/2012/8/8e/KAIST_20_Sept.JPG"></img></div>
-				<div style="clear:both;"></div>
+				<div style="clear:both;"></div></br>
-				<span id="little">bFMO PCR w/ various condtions</span>
+				<span id="little">bFMO PCR w/ various condtions</span></br>
 				</br>
@@ Line 518: / Line 1,003: @@
 		<section id="21">
 			<div class="date">September 21<sup>st</sup> 2012</div></br>
+			<div id="content_note" >
+				<span id="little">No Special Event!</span>
+				</br>
+			</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>
 		</section>
 		<section id="22">
@@ Line 607: / Line 1,098: @@
 				</br>
+</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">P1, PI construct </div>
+			<div id="content_note" >
+				<b>Results</b></br></br>
+				<span id="little">Full fragment for pAuto is overlapped. The right size(3794bp) fragments are gel extracted. </br></br>
+				</span>
+				<div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/4/41/120924-2.PNG"></img></div>
+				<div style="clear:both;"></div>
+			</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>
 			<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>
@@ Line 620: / Line 1,121: @@
 		<section id="25">
 			<div class="date">September 25<sup>th</sup> 2012</div></br>
+<div id="content_note" >
+				<span id="little">No Special Event!</span>
+				</br>
+			</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>
 		</section>
 		<section id="26">
 			<div class="date">September 26<sup>th</sup> 2012</div></br>
+				<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><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:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/>　Flip Flop<img style="height:3px;width:269px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
+			<div class="note-title">Full pAuto and pAutoSimple Overlapping PCR</div>
+			<div id="content_note" >
+				<b>Results</b></br></br>
+				<div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/2/21/120926-1.PNG"></img></div>
+				<div style="clear:both;"></div>
+				</br></br>
+				<b>Discussion</b></br></br>
+				<span id="little">The band intensity is too weak.</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"> pAutoIntegrase Overlapping PCR</div>
+			<div id="content_note" >
+				<b>Results</b></br></br>
+				<div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/0/04/120926-2-1.PNG"></img></div>
+				<div style="clear:both;"></div>
+				<span id="little"></br>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. </br></br>
+				</span>
+				<div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/0/06/120926-2-2.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"> Template preparation of terminator</div>
+				<div id="content_note" >
+				<b>Results</b></br></br>
+				<div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/f/fd/120926-3.PNG"></img></div>
+				<div style="clear:both;"></div>
+				<span id="little"></br>Terminator template for OE pcr is re-amplified with pfu-X DNA polymerase. The products are pcr purified. </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="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>
 		</section>
 		<section id="27">
-			<div class="date">September 27<sup>th</sup> 2012</div></br>
+<div class="date">September 27<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">Pre-culture of Gibson F1, F2 colonies</div>
+			<div id="content_note" >
+		<b>Results</b></br></br>
+				<span id="little">We checked that there were several colonies on the plate. 10 colonies for each fragment were picked, and culture into 3mL LB media containing 30ul Kanamycin.</br></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>
 		</section>
 		<section id="28">
 			<div class="date">September 28<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">Vector preparation and PCR</div>
+			<div id="content_note" >
+		<b>Results</b></br></br>
+				<span id="little">For each cultured cells, vectors were mini-prepped, and pcr was done with proper primers (F1 : 1201 F, 1197R, F2 : 1198F, 1516R)</br></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>
 		</section>
 		<section id="29">
 			<div class="date">September 29<sup>th</sup> 2012</div></br>
+			<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><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:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/>　Flip Flop<img style="height:3px;width:269px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
+			<div class="note-title">Template preparation of bFMO (for pAuto and pAutoSimple)</div>
+			<div id="content_note" >
+				<b>Results</b></br></br>
+				<div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/f/f8/120929-1.PNG"></img></div>
+				<div style="clear:both;"></div>
+				<span id="little"></br>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.
+</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"> Overlapping extension PCR Gel Extraction</div>
+			<div id="content_note" >
+				<b>Results</b></br></br>
+				<div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/7/7d/120930.PNG"></img></div>
+				<div style="clear:both;"></div>
+			</div>
+				</br></br>
+			</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>
 		</section>
 		<section id="30">
 			<div class="date">September 30<sup>th</sup> 2012</div></br>
-		</section>
+			<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><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:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/>　Flip Flop<img style="height:3px;width:269px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
-		<section id="31">
+			<div class="note-title">Template vector preparation – double digestion check with EcoRI and PstI</div>
-			<div class="date">September 31<sup>st</sup> 2012</div></br>
+			<div id="content_note" >
+				<b>Results</b></br></br>
+				<div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/0/0a/120930-..PNG"></img></div>
+				<div style="clear:both;"></div>
+				<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/d/d8/120829_table.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>
+			</span>
+			<div id="content_note" >
+				<div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/d/d8/120829_table.PNG"></img></div>
+				<div style="clear:both;"></div>
+			</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="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>
 		</section>

Team:KAIST Korea/Notebook Labnote/2012 9

From 2012.igem.org

Latest revision as of 03:59, 27 October 2012

Labnote