Team:CityU HK/notebook/lablog

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</head>
</head>
<body>
<body>
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<br><br>
 
-
<center> <font size = "6" > <b> TesA module's lablog </b> </font> </center><br>
 
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            <div class="wrapper">
+
<div id='cssmenu'>
 +
<ul>
 +
  <li><a href='https://2014.igem.org/Team:CityU_HK/notebook/lablog'><span>FadD & FadL Module</span></a></li>
 +
  <li><a href='https://2014.igem.org/Team:CityU_HK/notebook/lablog_2'><span>'TesA Module</span></a></li>
 +
  <li class='last'><a href='https://2014.igem.org/Team:CityU_HK/notebook/lablog_3'><span>Desaturase Module</span></a></li>
 +
</ul>
 +
</div>
 +
 
 +
<div><img src="https://static.igem.org/mediawiki/2014/e/ee/CityU_HK_lablog.png" width="100%"></div>
 +
 
 +
<div class="container"><div class="row"><div class="col-md-12">
 +
<div class="arrow-down" id="arrow2"></div>
 +
</div></div></div>
 +
 
 +
<h1 id="title">FadD & FadL Module's Lablog</h1>
 +
 
 +
<!-- START -->
 +
    <div class="wrapper">
                 <div id="st-accordion" class="st-accordion">
                 <div id="st-accordion" class="st-accordion">
 +
                     <ul>
                     <ul>
                                                
                                                
                         <li>
                         <li>
-
                             <a href="#">July<span class="st-arrow">Open or Close</span></a>
+
                             <a href="#">July - Week 4 (20/7~26/7)<span class="st-arrow">Open or Close</span></a>
                             <div class="st-content">
                             <div class="st-content">
-
                                <h2 class="title"><b>Week 3 (13/7~19/7)</b></h2>
+
                                 <p><b>-</b> PCR amplification of fadD, fadL inserts</p>
-
                                <p><b>-</b> Primer design for leaderless tesA for Fit-coli (‘tesA) & New biobrick (‘tesA BB)</p>
+
                                 <p><b>-</b> Purification of fadD, fadL PCR products</p>
-
                                <h2 class="title"><b>Week 4 (20/7~26/7)</b></h2>
+
                                 <p><b>-</b> LB plate preparation with chloramphenicol (34ug/ml) for selection of pSB1C3 clones</p>
-
                                 <p><b>-</b> PCR of ‘tesA and ‘tesA BB</p>
+
                                 <p><b>-</b> DH5α competent cells are transformed with BBa_B0030 and BBa_R0040 separately by heat shock</p>
-
                                 <p><b>-</b> PCR purification of ‘tesA and ‘tesA BB</p>
+
                                 <p><b>-</b> 1st Extraction of BBa_B0030, BBa_R0040 by miniprep<br> -> low product yield of BBa_R0040</p>
-
                                 <p><b>-</b> Prepare LB agar plates(‘tesA BB)</p>
+
                                 <p><b>-</b> Extraction of BBa_R0040, by miniprep<br> -> low product yield</p>
-
                                <h2 class="title"><b>Week 5 (27/7~31/7)</b></h2>
+
                                 <p><b>-</b> New batch of BBa_R0040 transformants are prepared in LB broth</p>
-
                                 <p><b>-</b> Digestion of ‘tesA BB and pSB1C3 plasmid with EcoRI & PstI restriction enzymes</p>
+
                         
-
                                 <p><b>-</b> PCR purification for digested ‘tesA BB</p>
+
                             </div>  
-
                                <p><b>-</b> Sub-cloning of ‘tesA BB into pSB1C3 plasmid</p>
+
-
                                 <p><b>-</b> Transformation of ‘tesA BB into W3110 E.coli</p>
+
-
                                 <p><b>-</b> Pick colonies of the transformed E. coli with ‘tesA BB</p>
+
-
                             </div>
+
                         </li>
                         </li>
-
                         <li>
+
<li>
 +
                            <a href="#">July - Week 5 (27/7~2/8)<span class="st-arrow">Open or Close</span></a>
 +
                            <div class="st-content">
 +
                                <p><b>-</b> Extraction of BBa_R0040 were performed with plasmid miniprep kit</p>
 +
                                <p><b>-</b> Restriction digestion were performed on:
 +
<br>  fadL, fadD inserts with XbaI and PstI
 +
<br>  BBa_R0040, BBa_B0030 with SpeI and PstI
 +
<br>Results: No band was observed for BBa_B0030 after digestion
 +
<br> -> Gel photo showed the extracted BBa_B0030 DNA was contaminated with RNA leading to overestimation of BBa_B0030 concentration for restriction digestion
 +
</p>
 +
                                <p><b>-</b> Purification was performed on the digested <i>fadL</i> and <i>fadD</i> inserts and BBa_R0040 products</p>
 +
                                <p><b>-</b> Ligation of digested <i>fadD</i> insert into BBa_B0030 vector was performed</p>
 +
                                <p><b>-</b> DH5α competent cell was transformed with pSB1C3_ R0040-fadL by heat shock protocol</p>
 +
                                <p><b>-</b> BBa_B0030 was performed again with plasmid DNA miniprep kit, the product was treated with additional RNase A</p>
 +
                                <p><b>-</b> Restriction digestion of BBa_B0030 was performed with SpeI and PstI</p>
 +
                                <p><b>-</b> Digested BBa_B0030 was purified with PCR purification kit</p>
 +
                                <p><b>-</b> <i>fadL</i> insert was ligated into BBa_R0040 vector</p>
 +
                                <p><b>-</b> LB plate with chloramphenicol (34 µg/mL) was prepared</p>
 +
                            </div> 
 +
                         </li>
 +
 
 +
<li>
                             <a href="#">Early August<span class="st-arrow">Open or Close</span></a>
                             <a href="#">Early August<span class="st-arrow">Open or Close</span></a>
                             <div class="st-content">
                             <div class="st-content">
-
                                 <h2 class="title"><b>Week 1 (1/8~2/8)</b></h2>
+
                                 <h2 class="title"><b>Week 1 (3/8~9/8)</b></h2>
-
                                 <p><b>-</b> Cell lysis of picked E. coli with ‘tesA BB</p>
+
                                 <p><b>-</b> DH5α competent cells were transformed with pSB1C3_ B0030-fadD by heat shock protocol</p>
-
                                 <p><b>-</b> Colonies PCR of ‘tesA BB by using VF2 & VR primer</p>
+
                                 <p><b>-</b> pSB1C3_ R0040-<i>fadL</i> clones was screened with colony PCR method                  <br> -> non-specific bands were observed</p>
-
                                 <p><b>-</b> Send the ‘tesA BB plasmid for sequencing</p>
+
                                <p><b>-</b> pSB1C3_ B0030-<i>fadD</i> clones were screened by colony PCR method                    <br> -> non-specific bands were observed</p>
 +
                                 <p><b>-</b> Repeated colony PCR analysis on the pSB1C3_ R0040-fadL clones                    <br> -> Bands with expected size were observed in two clones and sent for DNA sequencing</p>
 +
                                <p><b>-</b> pSB1C3_ B0030-<i>fadD</i> clones were screening again with colony PCR method                    <br> -> DNA Bands with expected size were observed in 6 clones and sent for DNA sequencing</p>
-
                                 <h2 class="title"><b>Week 2 (3/8~9/8)</b></h2>
+
                                 <h2 class="title"><b>Week 2 (10/8~16/8)</b></h2>
-
                                 <p><b>-</b> 2nd PCR of ‘tesA <br>∵ The first PCR product of ‘tesA was degraded over a long period of storage time</p>
+
                                 <p><b>-</b> Sequencing results show that the transformants contained errors in DNA sequences<br> -> Missing one to two base pairs in the <i>fadL</i> gene in the pSB1C3_ R0040-fadL clones<br> -> Missing the ribosomal binding site in the pSB1C3_ B0030-<i>fadD</i> clones</p>
-
                                 <p><b>-</b> PCR purification of ‘tesA PCR product</p>
+
                                 <p><b>-</b> Repeat screening for pSB1C3_ R0040-<i>fadL</i> clones were performed with the colony PCR method<br> -> Bands with expected size were observed in 4 clones and sent for DNA sequencing</p>
-
                                 <p><b>-</b> 1st Digestion of ‘tesA by using XbaI & PstI restriction enzyme<br>-> After running gel, no fragment showed in pilot ( other plasmid with the Xbal & Pstl respectively) , could not determine whether the digestion was success or not</p>
+
                                 <p><b>-</b> PCR amplification of <i>fadD</i>, <i>fadL</i> genes with higher annealing temperature</p>
-
                                 <p><b>-</b> 2nd digestion of ‘tesA by using XbaI & PstI restriction enzyme with adding one more control pilot that is only plasmid with no enzyme<br>-> Still no fragment presented in pilot, we wonder whether the pilot plasmid or the restriction enzyme had problem</p>
+
                                 <p><b>-</b> Purification of <i>fadD</i>, <i>fadL</i> PCR products</p>
-
                                 <p><b>-</b> 3rd digestion of lac promoter in pSB1C3 plasmid & pbad promoter in pSB1C3 plasmid by using EcoRI, XbaI, SpeI & PstI restriction enzyme<br>-> All of the restriction enzyme worked normally, but the pbad promoter in pSB1C3 plasmid was degraded</p>
+
                                 <p><b>-</b> Double restriction digestion on <i>fadL</i>, <i>fadD</i> PCR products with XbaI and PstI</p>
-
                                 <p><b>-</b> 4th digestion of ‘tesA by using XbaI & PstI restriction enzyme</p>
+
                                <p><b>-</b> Extraction of BBa_B0034 (biobricks with the ribosomal binding site) plasmid by plasmid DNA miniprep kit</p>
-
                                 <p><b>-</b> Sub-cloning of‘tesA into rbs in pSB1C3 plasmid (Part: BBa_B0030)</p>
+
                                 <p><b>-</b> Extraction of BBa_R0040 plasmid with plasmid DNA miniprep kit</p>
-
                                  
+
                                 <p><b>-</b> Double restriction digestion on BBa_R0040 plasmid with SpeI and PstI</p>
-
                             </div>
+
                                 <p><b>-</b> Purification of the digested BBa_R0040 plasmid</p>
 +
                             </div>
                         </li>
                         </li>
-
                        <li>
+
<li>
                             <a href="#">Late August<span class="st-arrow">Open or Close</span></a>
                             <a href="#">Late August<span class="st-arrow">Open or Close</span></a>
                             <div class="st-content">
                             <div class="st-content">
-
                                 <h2 class="title"><b>Week 3 (10/8~16/8)</b></h2>
+
                                 <h2 class="title"><b>Week 3 (17/8~23/8)</b></h2>
-
                                 <p><b>-</b> 1st Transformation of ‘tesA with rbs (BBa_B0030) into W3110 E.coli</p>
+
                                 <p><b>-</b> Sequencing results showed errors in DNA sequences in the clones prepared last week<br> -> Missing one to two base pairs in the <i>fadL</i> gene in the pSB1C3_ R0040-fadL plasmid</p>
-
                                 <p><b>-</b> Pick colonies of the transformed E. coli with ‘tesA & rbs (BBa_B0030)</p>
+
                                <p><b>-</b> Ligation was performed on the <i>fadL</i> insert and the BBa_R0040 vector</p>
-
                                 <p><b>-</b> Send the‘tesA with rbs (BBa_B0030) plasmid for sequencing<br>∵ The sequencing result showed the coding error of the rbs<br> -> Used BBa_B0034 rbs in pSB1C3 plasmid to replace BBa_B0030 rbs for 2nd cloning </p>
+
                                <p><b>-</b> DH5α competent cell was transformed with the pSB1C3_ R0040-fadL plasmid by the heat shock protocol</p>
-
                               
+
                                 <p><b>-</b> Double restriction digestion on BBa_B0034 by SpeI and PstI </p>
 +
                                <p><b>-</b> Purification of the digested BBa_B0034 plasmid</p>
 +
                                <p><b>-</b> Ligation were performed on the <i>fadD</i> insert and the BBa_B0034 vector</p>
 +
                                <p><b>-</b> LB plate with ampicillin (100 µg/mL) were prepared for the selection of pSB1A2 clones</p>
 +
                                 <p><b>-</b> DH5α competent cells were transformed with pSB1A2_ B0034-<i>fadD</i> by the heat shock protocol</p>
 +
                                <p><b>-</b> Screening for pSB1C3_ R0040-<i>fadL</i> clones were done by the colony PCR method                <br> -> DNA bands with expected size were observed in only one clone and sent for DNA sequencing</p>
-
                                 <h2 class="title"><b>Week 4 (17/8~23/8)</b></h2>
+
                                 <h2 class="title"><b>Week 4 (24/8~30/8)</b></h2>
-
                                <p><b>-</b> 3rd PCR of tesA’ <br>∵ The 2nd PCR product of ‘tesA was used up</p>
+
                                 <p><b>-</b> Screening on the pSB1A2_ B0034-fadD clones were done by the colony PCR method <br> -> DNA bands with expected size were observed in 4 clones and sent for DNA sequencing</p>
-
                                <p><b>-</b> PCR purification of ‘tesA PCR product</p>
+
                                 <p><b>-</b> PCR amplification of the <i>fadD</i>, <i>fadL</i> inserts were performed at higher annealing temperature</p>
-
                                <p><b>-</b> 5th digestion of ‘tesA by using XbaI and PstI restriction enzyme</p>
+
                                 <p><b>-</b> LB plates with ampicillin (100 µg/mL) were prepared</p>
-
                                <p><b>-</b> Sub-cloning of ‘tesA into rbs in pSB1A2 plasmid (Part: pSB1A2- BBa_B0034)</p>
+
                             
-
                                <p><b>-</b> Miniprep of pbad promoter (Part: pSB1C3-BBa_I13453)</p>
+
                             </div>
-
                                <p><b>-</b> 2nd tramsformation of ‘tesA with rbs (BBa_B0034) into W3110 E.coli</p>
+
-
                                <p><b>-</b> 1st digestion of pbad promoter (BBa_I13453) by using SpeI & PstI restriction enzyme</p>
+
-
 
+
-
                                <h2 class="title"><b>Week 5 (24/8~30/8)</b></h2>
+
-
                                 <p><b>-</b> Pick colonies of the transformed E. coli with ‘tesA & rbs (BBa_B0034)</p>
+
-
                                <p><b>-</b> Cell lysis of the picked E. coli with ‘tesA & rbs (BBa_B0034)</p>
+
-
                                <p><b>-</b> Colonies PCR of ‘tesA & rbs (BBa_B0034) by using VF2 & VR primer</p>
+
-
                                <p><b>-</b> Send the‘tesA with rbs (BBA-B0034) plasmid for sequencing</p>
+
-
                                 <p><b>-</b> Miniprep of ‘tesA with rbs (BBa_B0034)</p>
+
-
                                <p><b>-</b> 1st digestion of ‘tesA with rbs (BBa_B0034) by using XbaI & PstI restriction enzyme</p>
+
-
                                 <p><b>-</b> Gel purification of digested ‘tesA with rbs (BBa_B0034)</p>
+
-
                                <p><b>-</b> PCR purification of pbad promoter (BBa_I13453) digested in week 4</p>
+
-
                             </div>
+
                         </li>
                         </li>
-
                                                                                                                           
+
 
-
                                             
+
<li>
 +
                            <a href="#">September - Week 1 (31/8~6/9)<span class="st-arrow">Open or Close</span></a>
 +
                            <div class="st-content">
 +
                                <p><b>-</b> Sequencing results:
 +
<br>  The identity of the DNA insert in pSB1C3_ R0040-fadL clone was confirmed by DNA sequencing.
 +
<br>  Changes in DNA sequence were found in the fadD gene in pSB1A2_ B0034-fadD clones
 +
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Latest revision as of 16:54, 17 October 2014

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FadD & FadL Module's Lablog

  • July - Week 4 (20/7~26/7)Open or Close

    - PCR amplification of fadD, fadL inserts

    - Purification of fadD, fadL PCR products

    - LB plate preparation with chloramphenicol (34ug/ml) for selection of pSB1C3 clones

    - DH5α competent cells are transformed with BBa_B0030 and BBa_R0040 separately by heat shock

    - 1st Extraction of BBa_B0030, BBa_R0040 by miniprep
    -> low product yield of BBa_R0040

    - Extraction of BBa_R0040, by miniprep
    -> low product yield

    - New batch of BBa_R0040 transformants are prepared in LB broth

  • July - Week 5 (27/7~2/8)Open or Close

    - Extraction of BBa_R0040 were performed with plasmid miniprep kit

    - Restriction digestion were performed on:
    fadL, fadD inserts with XbaI and PstI
    BBa_R0040, BBa_B0030 with SpeI and PstI
    Results: No band was observed for BBa_B0030 after digestion
    -> Gel photo showed the extracted BBa_B0030 DNA was contaminated with RNA leading to overestimation of BBa_B0030 concentration for restriction digestion

    - Purification was performed on the digested fadL and fadD inserts and BBa_R0040 products

    - Ligation of digested fadD insert into BBa_B0030 vector was performed

    - DH5α competent cell was transformed with pSB1C3_ R0040-fadL by heat shock protocol

    - BBa_B0030 was performed again with plasmid DNA miniprep kit, the product was treated with additional RNase A

    - Restriction digestion of BBa_B0030 was performed with SpeI and PstI

    - Digested BBa_B0030 was purified with PCR purification kit

    - fadL insert was ligated into BBa_R0040 vector

    - LB plate with chloramphenicol (34 µg/mL) was prepared

  • Early AugustOpen or Close

    Week 1 (3/8~9/8)

    - DH5α competent cells were transformed with pSB1C3_ B0030-fadD by heat shock protocol

    - pSB1C3_ R0040-fadL clones was screened with colony PCR method
    -> non-specific bands were observed

    - pSB1C3_ B0030-fadD clones were screened by colony PCR method
    -> non-specific bands were observed

    - Repeated colony PCR analysis on the pSB1C3_ R0040-fadL clones
    -> Bands with expected size were observed in two clones and sent for DNA sequencing

    - pSB1C3_ B0030-fadD clones were screening again with colony PCR method
    -> DNA Bands with expected size were observed in 6 clones and sent for DNA sequencing

    Week 2 (10/8~16/8)

    - Sequencing results show that the transformants contained errors in DNA sequences
    -> Missing one to two base pairs in the fadL gene in the pSB1C3_ R0040-fadL clones
    -> Missing the ribosomal binding site in the pSB1C3_ B0030-fadD clones

    - Repeat screening for pSB1C3_ R0040-fadL clones were performed with the colony PCR method
    -> Bands with expected size were observed in 4 clones and sent for DNA sequencing

    - PCR amplification of fadD, fadL genes with higher annealing temperature

    - Purification of fadD, fadL PCR products

    - Double restriction digestion on fadL, fadD PCR products with XbaI and PstI

    - Extraction of BBa_B0034 (biobricks with the ribosomal binding site) plasmid by plasmid DNA miniprep kit

    - Extraction of BBa_R0040 plasmid with plasmid DNA miniprep kit

    - Double restriction digestion on BBa_R0040 plasmid with SpeI and PstI

    - Purification of the digested BBa_R0040 plasmid

  • Late AugustOpen or Close

    Week 3 (17/8~23/8)

    - Sequencing results showed errors in DNA sequences in the clones prepared last week
    -> Missing one to two base pairs in the fadL gene in the pSB1C3_ R0040-fadL plasmid

    - Ligation was performed on the fadL insert and the BBa_R0040 vector

    - DH5α competent cell was transformed with the pSB1C3_ R0040-fadL plasmid by the heat shock protocol

    - Double restriction digestion on BBa_B0034 by SpeI and PstI

    - Purification of the digested BBa_B0034 plasmid

    - Ligation were performed on the fadD insert and the BBa_B0034 vector

    - LB plate with ampicillin (100 µg/mL) were prepared for the selection of pSB1A2 clones

    - DH5α competent cells were transformed with pSB1A2_ B0034-fadD by the heat shock protocol

    - Screening for pSB1C3_ R0040-fadL clones were done by the colony PCR method
    -> DNA bands with expected size were observed in only one clone and sent for DNA sequencing

    Week 4 (24/8~30/8)

    - Screening on the pSB1A2_ B0034-fadD clones were done by the colony PCR method
    -> DNA bands with expected size were observed in 4 clones and sent for DNA sequencing

    - PCR amplification of the fadD, fadL inserts were performed at higher annealing temperature

    - LB plates with ampicillin (100 µg/mL) were prepared

  • September - Week 1 (31/8~6/9)Open or Close

    - Sequencing results:
    The identity of the DNA insert in pSB1C3_ R0040-fadL clone was confirmed by DNA sequencing.
    Changes in DNA sequence were found in the fadD gene in pSB1A2_ B0034-fadD clones