Team:CityU HK/notebook/lablog

From 2014.igem.org

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<center> <font size = "6" > <b> FadD & FadL module's lablog </b> </font> </center><br>
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<center> <font size = "6" > <b> TesA module's lablog </b> </font> </center><br>
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<div class="wrapper">
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            <div class="wrapper">
                 <div id="st-accordion" class="st-accordion">
                 <div id="st-accordion" class="st-accordion">
                     <ul>
                     <ul>
                                                
                                                
                         <li>
                         <li>
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                             <a href="#">July - Week 4 (20/7~26/7)<span class="st-arrow">Open or Close</span></a>
+
                             <a href="#">July<span class="st-arrow">Open or Close</span></a>
                             <div class="st-content">
                             <div class="st-content">
-
                                 <p><b>-</b> PCR amplification of fadD, fadL inserts</p>
+
                                <h2 class="title"><b>Week 3 (13/7~19/7)</b></h2>
-
                                 <p><b>-</b> Purification of fadD, fadL PCR products</p>
+
                                <p><b>-</b> Primer design for leaderless tesA for Fit-coli (‘tesA) & New biobrick (‘tesA BB)</p>
-
                                 <p><b>-</b> LB plate preparation with chloramphenicol (34ug/ml) for selection of pSB1C3 clones</p>
+
                                <h2 class="title"><b>Week 4 (20/7~26/7)</b></h2>
-
                                 <p><b>-</b> DH5α competent cells are transformed with BBa_B0030 and BBa_R0040 separately by heat shock</p>
+
                                 <p><b>-</b> PCR 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> PCR purification of ‘tesA and ‘tesA BB</p>
-
                                 <p><b>-</b> Extraction of BBa_R0040, by miniprep<br> -> low product yield</p>
+
                                 <p><b>-</b> Prepare LB agar plates(‘tesA BB)</p>
-
                                 <p><b>-</b> New batch of BBa_R0040 transformants are prepared in LB broth</p>
+
                                <h2 class="title"><b>Week 5 (27/7~31/7)</b></h2>
-
                         
+
                                 <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>
 +
                                <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>
                             </div>
                         </li>
                         </li>
-
<li>
+
                        <li>
-
                             <a href="#">July - Week 5 (27/7~2/8)<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">
-
                                 <p><b>-</b> Extraction of BBa_R0040 by miniprep</p>
+
                                <h2 class="title"><b>Week 1 (1/8~2/8)</b></h2>
-
                                 <p><b>-</b> Restriction digestion of fadL, fadD inserts by XbaI and PstI  & BBa_R0040, BBa_B0030 (rbs) by SpeI and PstI <br> -> No band is observed for BBa_B0030 after digestion <br> -> Gel photo showing extracted BBa_B0030 is contaminated with RNA <br> -> Lead to overestimation of BBa_B0030 concentration for digestion</p>
+
                                 <p><b>-</b> Cell lysis of picked E. coli with ‘tesA BB</p>
-
                                 <p><b>-</b> Purification of digested fadL, fadD inserts, BBa_R0040 products</p>
+
                                 <p><b>-</b> Colonies PCR of ‘tesA BB by using VF2 & VR primer</p>
-
                                 <p><b>-</b> Ligation of digested fadD insert into BBa_B0030 vector</p>
+
                                <p><b>-</b> Send the ‘tesA BB plasmid for sequencing</p>
-
                                 <p><b>-</b> DH5α competent cell is transformed with pSB1C3_ R0040-fadL by heat shock</p>
+
 
-
                                 <p><b>-</b> Repeat extraction of BBa_B0030 by miniprep, product is treated with RNase A</p>
+
                                 <h2 class="title"><b>Week 2 (3/8~9/8)</b></h2>
-
                                 <p><b>-</b> Restriction digestion of Bba_B0030 by SpeI and PstI</p>
+
                                 <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> Purification of digested Bba_B0030</p>
+
                                 <p><b>-</b> PCR purification of ‘tesA PCR product</p>
-
                                 <p><b>-</b> Ligation of fadL insert into BBa_R0040 vector</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> LB plate preparation with Chloramphenicol (34ug/ml)</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> 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> 4th digestion of ‘tesA by using XbaI & PstI restriction enzyme</p>
 +
                                 <p><b>-</b> Sub-cloning of‘tesA into rbs in pSB1C3 plasmid (Part: BBa_B0030)</p>
 +
                               
                             </div>
                             </div>
                         </li>
                         </li>
 +
                        <li>
 +
                            <a href="#">Late August<span class="st-arrow">Open or Close</span></a>
 +
                            <div class="st-content">
 +
                                <h2 class="title"><b>Week 3 (10/8~16/8)</b></h2>
 +
                                <p><b>-</b> 1st Transformation of ‘tesA with rbs (BBa_B0030) into W3110 E.coli</p>
 +
                                <p><b>-</b> Pick colonies of the transformed E. coli with ‘tesA & rbs (BBa_B0030)</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>
 +
                               
 +
                                <h2 class="title"><b>Week 4 (17/8~23/8)</b></h2>
 +
                                <p><b>-</b> 3rd PCR of tesA’ <br>∵ The 2nd PCR product of ‘tesA was used up</p>
 +
                                <p><b>-</b> PCR purification of ‘tesA PCR product</p>
 +
                                <p><b>-</b> 5th digestion of ‘tesA by using XbaI and PstI restriction enzyme</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>
 +
                                <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>
-
                    </ul>
+
                                <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>
 +
                                                                                                                           
 +
                                             
 +
                    </ul>
                 </div>
                 </div>
             </div>
             </div>
 +
 +
 +
 +
             
<br><br><br>
<br><br><br>

Revision as of 07:35, 17 September 2014

Bootstrap 101 Template



TesA module's lablog

  • JulyOpen or Close

    Week 3 (13/7~19/7)

    - Primer design for leaderless tesA for Fit-coli (‘tesA) & New biobrick (‘tesA BB)

    Week 4 (20/7~26/7)

    - PCR of ‘tesA and ‘tesA BB

    - PCR purification of ‘tesA and ‘tesA BB

    - Prepare LB agar plates(‘tesA BB)

    Week 5 (27/7~31/7)

    - Digestion of ‘tesA BB and pSB1C3 plasmid with EcoRI & PstI restriction enzymes

    - PCR purification for digested ‘tesA BB

    - Sub-cloning of ‘tesA BB into pSB1C3 plasmid

    - Transformation of ‘tesA BB into W3110 E.coli

    - Pick colonies of the transformed E. coli with ‘tesA BB

  • Early AugustOpen or Close

    Week 1 (1/8~2/8)

    - Cell lysis of picked E. coli with ‘tesA BB

    - Colonies PCR of ‘tesA BB by using VF2 & VR primer

    - Send the ‘tesA BB plasmid for sequencing

    Week 2 (3/8~9/8)

    - 2nd PCR of ‘tesA
    ∵ The first PCR product of ‘tesA was degraded over a long period of storage time

    - PCR purification of ‘tesA PCR product

    - 1st Digestion of ‘tesA by using XbaI & PstI restriction enzyme
    -> 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

    - 2nd digestion of ‘tesA by using XbaI & PstI restriction enzyme with adding one more control pilot that is only plasmid with no enzyme
    -> Still no fragment presented in pilot, we wonder whether the pilot plasmid or the restriction enzyme had problem

    - 3rd digestion of lac promoter in pSB1C3 plasmid & pbad promoter in pSB1C3 plasmid by using EcoRI, XbaI, SpeI & PstI restriction enzyme
    -> All of the restriction enzyme worked normally, but the pbad promoter in pSB1C3 plasmid was degraded

    - 4th digestion of ‘tesA by using XbaI & PstI restriction enzyme

    - Sub-cloning of‘tesA into rbs in pSB1C3 plasmid (Part: BBa_B0030)

  • Late AugustOpen or Close

    Week 3 (10/8~16/8)

    - 1st Transformation of ‘tesA with rbs (BBa_B0030) into W3110 E.coli

    - Pick colonies of the transformed E. coli with ‘tesA & rbs (BBa_B0030)

    - Send the‘tesA with rbs (BBa_B0030) plasmid for sequencing
    ∵ The sequencing result showed the coding error of the rbs
    -> Used BBa_B0034 rbs in pSB1C3 plasmid to replace BBa_B0030 rbs for 2nd cloning

    Week 4 (17/8~23/8)

    - 3rd PCR of tesA’
    ∵ The 2nd PCR product of ‘tesA was used up

    - PCR purification of ‘tesA PCR product

    - 5th digestion of ‘tesA by using XbaI and PstI restriction enzyme

    - Sub-cloning of ‘tesA into rbs in pSB1A2 plasmid (Part: pSB1A2- BBa_B0034)

    - Miniprep of pbad promoter (Part: pSB1C3-BBa_I13453)

    - 2nd tramsformation of ‘tesA with rbs (BBa_B0034) into W3110 E.coli

    - 1st digestion of pbad promoter (BBa_I13453) by using SpeI & PstI restriction enzyme

    Week 5 (24/8~30/8)

    - Pick colonies of the transformed E. coli with ‘tesA & rbs (BBa_B0034)

    - Cell lysis of the picked E. coli with ‘tesA & rbs (BBa_B0034)

    - Colonies PCR of ‘tesA & rbs (BBa_B0034) by using VF2 & VR primer

    - Send the‘tesA with rbs (BBA-B0034) plasmid for sequencing

    - Miniprep of ‘tesA with rbs (BBa_B0034)

    - 1st digestion of ‘tesA with rbs (BBa_B0034) by using XbaI & PstI restriction enzyme

    - Gel purification of digested ‘tesA with rbs (BBa_B0034)

    - PCR purification of pbad promoter (BBa_I13453) digested in week 4




TesA module's lablog

  • JulyOpen or Close

    Week 3 (13/7~19/7)

    - Primer design for leaderless tesA for Fit-coli (‘tesA) & New biobrick (‘tesA BB)

    Week 4 (20/7~26/7)

    - PCR of ‘tesA and ‘tesA BB

    - PCR purification of ‘tesA and ‘tesA BB

    - Prepare LB agar plates(‘tesA BB)

    Week 5 (27/7~31/7)

    - Digestion of ‘tesA BB and pSB1C3 plasmid with EcoRI & PstI restriction enzymes

    - PCR purification for digested ‘tesA BB

    - Sub-cloning of ‘tesA BB into pSB1C3 plasmid

    - Transformation of ‘tesA BB into W3110 E.coli

    - Pick colonies of the transformed E. coli with ‘tesA BB

  • Early AugustOpen or Close

    Week 1 (1/8~2/8)

    - Cell lysis of picked E. coli with ‘tesA BB

    - Colonies PCR of ‘tesA BB by using VF2 & VR primer

    - Send the ‘tesA BB plasmid for sequencing

    Week 2 (3/8~9/8)

    - 2nd PCR of ‘tesA
    ∵ The first PCR product of ‘tesA was degraded over a long period of storage time

    - PCR purification of ‘tesA PCR product

    - 1st Digestion of ‘tesA by using XbaI & PstI restriction enzyme
    -> 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

    - 2nd digestion of ‘tesA by using XbaI & PstI restriction enzyme with adding one more control pilot that is only plasmid with no enzyme
    -> Still no fragment presented in pilot, we wonder whether the pilot plasmid or the restriction enzyme had problem

    - 3rd digestion of lac promoter in pSB1C3 plasmid & pbad promoter in pSB1C3 plasmid by using EcoRI, XbaI, SpeI & PstI restriction enzyme
    -> All of the restriction enzyme worked normally, but the pbad promoter in pSB1C3 plasmid was degraded

    - 4th digestion of ‘tesA by using XbaI & PstI restriction enzyme

    - Sub-cloning of‘tesA into rbs in pSB1C3 plasmid (Part: BBa_B0030)

  • Late AugustOpen or Close

    Week 3 (10/8~16/8)

    - 1st Transformation of ‘tesA with rbs (BBa_B0030) into W3110 E.coli

    - Pick colonies of the transformed E. coli with ‘tesA & rbs (BBa_B0030)

    - Send the‘tesA with rbs (BBa_B0030) plasmid for sequencing
    ∵ The sequencing result showed the coding error of the rbs
    -> Used BBa_B0034 rbs in pSB1C3 plasmid to replace BBa_B0030 rbs for 2nd cloning

    Week 4 (17/8~23/8)

    - 3rd PCR of tesA’
    ∵ The 2nd PCR product of ‘tesA was used up

    - PCR purification of ‘tesA PCR product

    - 5th digestion of ‘tesA by using XbaI and PstI restriction enzyme

    - Sub-cloning of ‘tesA into rbs in pSB1A2 plasmid (Part: pSB1A2- BBa_B0034)

    - Miniprep of pbad promoter (Part: pSB1C3-BBa_I13453)

    - 2nd tramsformation of ‘tesA with rbs (BBa_B0034) into W3110 E.coli

    - 1st digestion of pbad promoter (BBa_I13453) by using SpeI & PstI restriction enzyme

    Week 5 (24/8~30/8)

    - Pick colonies of the transformed E. coli with ‘tesA & rbs (BBa_B0034)

    - Cell lysis of the picked E. coli with ‘tesA & rbs (BBa_B0034)

    - Colonies PCR of ‘tesA & rbs (BBa_B0034) by using VF2 & VR primer

    - Send the‘tesA with rbs (BBA-B0034) plasmid for sequencing

    - Miniprep of ‘tesA with rbs (BBa_B0034)

    - 1st digestion of ‘tesA with rbs (BBa_B0034) by using XbaI & PstI restriction enzyme

    - Gel purification of digested ‘tesA with rbs (BBa_B0034)

    - PCR purification of pbad promoter (BBa_I13453) digested in week 4




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