Team:CityU HK/notebook/lablog
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- | <br> <br><br>< | + | <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"> | ||
+ | |||
+ | <ul> | ||
+ | |||
+ | <li> | ||
+ | <a href="#">July - Week 4 (20/7~26/7)<span class="st-arrow">Open or Close</span></a> | ||
+ | <div class="st-content"> | ||
+ | <p><b>-</b> PCR amplification of fadD, fadL inserts</p> | ||
+ | <p><b>-</b> Purification of fadD, fadL PCR products</p> | ||
+ | <p><b>-</b> LB plate preparation with chloramphenicol (34ug/ml) for selection of pSB1C3 clones</p> | ||
+ | <p><b>-</b> DH5α competent cells are transformed with BBa_B0030 and BBa_R0040 separately by heat shock</p> | ||
+ | <p><b>-</b> 1st Extraction of BBa_B0030, BBa_R0040 by miniprep<br> -> low product yield of BBa_R0040</p> | ||
+ | <p><b>-</b> Extraction of BBa_R0040, by miniprep<br> -> low product yield</p> | ||
+ | <p><b>-</b> New batch of BBa_R0040 transformants are prepared in LB broth</p> | ||
+ | |||
+ | </div> | ||
+ | </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> | ||
+ | <div class="st-content"> | ||
+ | <h2 class="title"><b>Week 1 (3/8~9/8)</b></h2> | ||
+ | <p><b>-</b> DH5α competent cells were transformed with pSB1C3_ B0030-fadD by heat shock protocol</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> 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 (10/8~16/8)</b></h2> | ||
+ | <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> 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> PCR amplification of <i>fadD</i>, <i>fadL</i> genes with higher annealing temperature</p> | ||
+ | <p><b>-</b> Purification of <i>fadD</i>, <i>fadL</i> PCR products</p> | ||
+ | <p><b>-</b> Double restriction digestion on <i>fadL</i>, <i>fadD</i> PCR products with XbaI and PstI</p> | ||
+ | <p><b>-</b> Extraction of BBa_B0034 (biobricks with the ribosomal binding site) plasmid by plasmid DNA miniprep kit</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> | ||
+ | <p><b>-</b> Purification of the digested BBa_R0040 plasmid</p> | ||
+ | </div> | ||
+ | </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 (17/8~23/8)</b></h2> | ||
+ | <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> Ligation was performed on the <i>fadL</i> insert and the BBa_R0040 vector</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 (24/8~30/8)</b></h2> | ||
+ | <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 amplification of the <i>fadD</i>, <i>fadL</i> inserts were performed at higher annealing temperature</p> | ||
+ | <p><b>-</b> LB plates with ampicillin (100 µg/mL) were prepared</p> | ||
+ | |||
+ | </div> | ||
+ | </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 | ||
+ | </p> | ||
+ | </div> | ||
+ | </li> | ||
+ | </ul> | ||
+ | </div> <!-- end of .st-accordion--> | ||
+ | </div> <!--end of wrapper--> | ||
- | < | + | <!-- END --> |
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+ | <!--==================== footer ===========================--> | ||
+ | <div id="footer" class="row"> | ||
+ | <div class="col-md-7"> | ||
+ | <h3>Sponsors</h3> | ||
+ | <a href="http://www.cityu.edu.hk/" target="_blank"><img src="https://static.igem.org/mediawiki/2014/9/9e/CityU_HK_cityu30-logo.png" width="95"></a> | ||
+ | <a href="http://www6.cityu.edu.hk/bhdbapp/deptweb/index.html" target="_blank"><img src="https://static.igem.org/mediawiki/2014/9/98/CityU_HK_bchlogo.png" width="80"></a> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/b/b1/CityU_HK_Invitrogen.png" width="100"> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/7/7a/CityU_HK_Neblogocol.png" width="135"> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/d/dc/CityU_HK_IDT-Logo.png" width="125"> | ||
+ | </div> <!-- end of col-md-7--> | ||
+ | <div class="col-md-5"> | ||
+ | <h3>Stay connected!</h3> | ||
+ | <p>email: <a href="mailto:cityuhk.igem@gmail.com">cityuhk.igem@gmail.com</a></p> | ||
+ | <a href="http://www.facebook.com/igem.2014CityU"><img src="https://static.igem.org/mediawiki/2014/3/31/CityU_HK_fb_logo.png" width="50" height="48"></a> | ||
+ | <a href="http://twitter.com/CityUHK_iGEM"><img src="http://ibo2014.org/wordpress/wp-content/themes/ibo2014/img/navbar/twitter_icon.png" width="49" height="51"></a> | ||
+ | <a href="#"><img src="https://static.igem.org/mediawiki/2014/1/12/CityU_HK_youtube_logo.png" width="52" height="50"></a> | ||
+ | </div> <!--end of col-md-5--> | ||
+ | </div> | ||
+ | <div class="row"> | ||
+ | <div id="copyright" class="col-md-12"> | ||
+ | <p>Copyright © iGEM CityU HK 2014. All Rights Reserved</p> | ||
+ | </div> | ||
+ | </div> | ||
+ | <!--end of footer--> | ||
</body> | </body> | ||
</html> | </html> |
Latest revision as of 16:54, 17 October 2014
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 sequencingWeek 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 sequencingWeek 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
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