Difference between revisions of "Team:Warwick/Project5"

 
(27 intermediate revisions by 3 users not shown)
Line 2: Line 2:
 
{{WarwickTimeline}}
 
{{WarwickTimeline}}
 
<html>
 
<html>
−
<br>
 
−
<br>
 
−
<br>
 
  
−
<!-- SLIDER
+
 
−
================================================== -->
+
−
<div id="ei-slider" class="ei-slider">
+
−
<ul class="ei-slider-large">
+
−
+
−
<li>
+
−
<a href="#link1"><img src="http://p1.pichost.me/i/61/1854415.jpg" alt="image10" class="responsiveslide"></a>
+
−
</li>
+
−
</ul>
+
−
+
−
</div>
+
−
<div class="minipause">
+
−
</div>
+
  
 
<!-- SUBHEADER
 
<!-- SUBHEADER
Line 53: Line 38:
 
<h2>Week 1</h2>
 
<h2>Week 1</h2>
 
<p>We brainstormed ideas of what we can do with the project, more specifically and also split into smaller groups of modellers and biologists (although we had daily meetings). We spent a lot of time coming up with and optimising DNA sequences for zinc fingers.</p>
 
<p>We brainstormed ideas of what we can do with the project, more specifically and also split into smaller groups of modellers and biologists (although we had daily meetings). We spent a lot of time coming up with and optimising DNA sequences for zinc fingers.</p>
−
<a href="#0" class="cd-read-more">Read more</a>
+
 
<span class="cd-date">Jun 29</span>
 
<span class="cd-date">Jun 29</span>
 
</div> <!-- cd-timeline-content -->
 
</div> <!-- cd-timeline-content -->
Line 65: Line 50:
 
<div class="cd-timeline-content">
 
<div class="cd-timeline-content">
 
<h2>Week 2</h2>
 
<h2>Week 2</h2>
−
<p>7/7/15
+
<p>
−
 
+
−
<br>We made 8 plates of streptomycin and chloramphenicol  ­ 3 plates of chloramphenicol ­ Cells made electrocompetent following lab protocol
+
−
 
+
−
<br>8/7/15
+
−
 
+
−
­ <br>Cells grew!
+
−
 
+
−
<br> <b>Cells transformed-</b> ­
+
  
 +
<br>07/07/2015:
  
−
<br>INP 8H psB1c3 5
+
- <br>Agar plates (with chloramphenicol and streptomycin) made.
  
−
<br>Ipp OmpA 6L psB1c4 5
+
- <br>Top 10 cells made electrocompetent.
  
−
<br>Zif 23 1G psB1c5 3
+
<br>08/07/2015:
  
−
<br>9/7/15  ­ <br>Met Miriam ­ phD presentation and explanation of cloning/ gibson assembly/pcr
+
- <br>Transformed Top 10 cells with INP, Lpp OmpA and Zif 23 from 2015 DNA
  
−
­ <br>4 minipreps  ­ Nanodrop results: J04450 ­ 55.1ng/l
+
Distribution Kit.
 +
<p><img src="https://static.igem.org/mediawiki/2015/0/01/Distribution_Kit_table_Lab_Book.png" height="60px" width="400px" border="1px"></p>
 +
<br>09/07/2015:
  
−
                              <br>INP ­ 10.8 ng/l
+
- <br>Mini-prepped cells from yesterday.
  
−
                            <br> C2001 ­ 16.1 ng/l
+
- <br>Nanodrop results for mini-prepped plasmids.
 +
<p><img src="https://static.igem.org/mediawiki/2015/7/7b/NanoDropResultsTableLab_BookiGEMWarwick.png" height="60px" width="400px" border="1px"></p>
 +
<br>Ran an electrophoresis gel with plasmids from the mini-prep.
  
−
                            <br>  LPPOMPA ­ 38.4 ng/l ­ Electrophoresis: Made 10% urea, 1% agarose gel ethidum gel
 
  
−
                          <br>Put plasmids from mini prep in gel, ran for 30 mins at 100V
 
−
 
−
­ (Left to right: Ladder, LPPOMPA, C2001, INP, J04450)
 
 
</p>
 
</p>
−
<a href="#0" class="cd-read-more">Read more</a>
+
 
<span class="cd-date">Jul 06</span>
 
<span class="cd-date">Jul 06</span>
 
</div> <!-- cd-timeline-content -->
 
</div> <!-- cd-timeline-content -->
Line 109: Line 87:
 
<div class="cd-timeline-content">
 
<div class="cd-timeline-content">
 
<h2>Week 3</h2>
 
<h2>Week 3</h2>
−
<p>We grew up MG1655 (Z1) cells.
+
<p>
−
<br> Put into 3ml LB, added 3l Strep and put in a shaking incubator.
+
 
−
<br> We also made competent cells.</p>
+
 
−
<a href="#0" class="cd-read-more">Read more</a>
+
­ <br>13/07/2015:
 +
 
 +
- <br>Grew up MG1655-Z1 cells (added Streptomycin).
 +
 
 +
<br>14/07/2015:
 +
 
 +
- <br>Made MG1655-Z1 cells competent.
 +
 
 +
 
 +
 
 +
</p>
 +
 
<span class="cd-date">Jul 13</span>
 
<span class="cd-date">Jul 13</span>
 
</div> <!-- cd-timeline-content -->
 
</div> <!-- cd-timeline-content -->
Line 126: Line 115:
 
<div class="cd-timeline-content">
 
<div class="cd-timeline-content">
 
<h2>Week 4</h2>
 
<h2>Week 4</h2>
−
<p>We ligated full construct g-block into Psb1C3 plasmid backbone.
+
<p>
−
<br>Diluted primers to 100mM, made set of 5mM primer tubes. ­ Transformed ligated plasmid into top 10 cells (still some plasmid left in the top shelf of
+
  
−
the freezer). ­ PCR: 5l 5mM forward primer
 
  
−
        <br> 5l 5mM reverse primer
+
<br>22/07/2015:
  
−
      <br>   0.5l of g­block
+
- <br>Ligated full construct gBlock into pSB1C3 plasmid backbone.
  
−
        <br> 25l of Q5 mastermix
+
<br>23/07/2015:
  
−
        <br> 14.5l of water
+
- <br>Transformed ligated plasmid into Top 10 cells.
−
<br> The ligated plasmid transformed cells did not grow overnight so were repeated. We undertook PCR for zf10, zf14 and zf2</p>
+
 
−
<a href="#0" class="cd-read-more">Read more</a>
+
- <br>Ran PCR of gBlocks.
 +
 
 +
<br>24/07/2015:
 +
 
 +
- <br>Transformed ligated plasmid into Top 10 cells again.
 +
 
 +
- <br>Ran PCR of BclA, INP and pgsA gBlocks.
 +
 
 +
- <br>Transformed more cells with ligated plasmid (using electroporation).
 +
 
 +
- <br>Plated transformed cells.
 +
 
 +
- <br>Ran PCR of sZF2, sZF10 and sZF14.
 +
 
 +
 
 +
</p>
 +
 
<span class="cd-date">Jul 20</span>
 
<span class="cd-date">Jul 20</span>
 
</div> <!-- cd-timeline-content -->
 
</div> <!-- cd-timeline-content -->
Line 153: Line 156:
 
<div class="cd-timeline-content">
 
<div class="cd-timeline-content">
 
<h2>Week 5</h2>
 
<h2>Week 5</h2>
−
<p>Both of the chemically transformed plates and electrotransformed plate grew over weekend.
+
<p>
−
<br>Lpp­ompA­zif268: colony PCR of clone 2 ok (inoculate culture to purify DNA ­ inoculate 2
+
 
 +
<br>27/07/2015:
 +
 
 +
- <br>Chemically transformed plates and electrotransformed plated grew over  
 +
 
 +
weekend.
 +
 
 +
<br>28/07/2015
 +
 
 +
- <br>Colony PCR of the 2 clones.
 +
 
 +
<br>29/07/2015
 +
 
 +
- <br>Conducted plasmid mini prep of bacterial cultures.
 +
 
 +
- <br>Ran gel electrophoresis of redone PCR.
 +
 
 +
- <br>Ran gel electrophoresis of mini prep restriction digestion.
 +
 
 +
- <br>Redoing PCR (of INP, sZF2, sZF10 and sZF14) with DMSO (at 0%, 3% and
 +
 
 +
6%).
 +
 
 +
- <br>PCR purification of BclA and  and pgsA.
 +
 
 +
<br>30/07/2015
 +
 
 +
- <br>Re-did PCR of INP, sZF10 and sZF14.
 +
 
 +
- <br>sZF14 returned positive (concentration of 31.8 ng/µl after purification).
 +
 
 +
- <br>Purified sZF2.
 +
 
 +
- <br>Re-doing PCR of INP and sZF10.
  
−
cultures, 4.5ml each. Miniprep them after making a glycerol stock).
+
<br>31/07/2015
−
<br>Conducted plasmid miniprep of bacterial culture. ­ Gel electrophoresis of redone PCR ­ Pgsa worked, zf2 (all others need to be repeated at
+
  
−
a later stage). ­ <br>Running gel of miniprep restriction digestion. ­ Re­doing PCR: INP, zf2, zf14 (67oC). zf10 (69oC) ­ with DMSO (0%, 3%, 6%). ­<br> PCR purification of BCLA and PGSA (nanodrop returned negative, redoing). ­ Redoing PCR of INP, zf2, zf10, zf14.
+
- <br>Gel of sZF10 and INP PCR product returned negative.  
−
<br>Redid PCR of INP, zf10 and zf14. ­ zf14 returned positive, purified (31.8 ng/l. ­ Redoing PCR of INP and zf10. ­ Purified zf2, nanodropped.
+
−
<br>Gel of zf10 and INP returned negative.
+
−
<br>Redoing PCR with 66oC and 680C annealing temperatures.  
+
  
−
<br>Restriction digestion protocol: 5l cutsmart 10x
+
- <br>Re-doing PCR with 66 ̊ C and 68 ̊ C annealing temperatures.  
−
                                                            1l DNA
+
−
                                                            10 units enzyme each
+
−
                                                            make up to 50l water
+
−
<br>For Nde1-Age1-HF, incubate at 370C for 5-15 minutes.
+
−
<br>Diluted INP g-block and ZF10 g-block by a factor of 10.
+
  
−
<br>Ran gradient PCR of INP and ZF10 (made 12 tubes of 20l).
+
- <br>Ran gradient PCR of sZF10 and INP.  
  
 +
- <br>Did restriction digestion of BclA, pgsA and Lpp OmpA
 
</p>
 
</p>
−
<a href="#0" class="cd-read-more">Read more</a>
+
 
<span class="cd-date">Jul 27</span>
 
<span class="cd-date">Jul 27</span>
 
</div> <!-- cd-timeline-content -->
 
</div> <!-- cd-timeline-content -->
Line 187: Line 215:
 
<h2>Week 6</h2>
 
<h2>Week 6</h2>
 
<p>
 
<p>
−
<br>3/8/15
+
<br>03/08/2015:
  
−
<br>Redoing restriction digest of LPP-OMPA
+
- <br>Re-did restriction digestion of Lpp OmpA.
−
<br>Running gel from Friday (last time = 6.1 ng/l, issue with plasmid?)
+
−
<br>Plasmid concentration only 5.9ng/l, running ligation anyway.
+
−
<br>20l ligation reaction (full details in book).
+
  
−
<br>4/8/15
+
- <br>Ligated BclA and pgsA into pSB1C3.
  
−
<br>Transforming ligated plasmid into mg1655 cells.
+
<br>04/08/2015:
−
<br>PCR purify INP.
+
−
<br>Restriction digestion of INP and LPP-OMPA.
+
−
<br>Diluted full construct g-block 10x.
+
−
<br>Gradient PCR of LPP-OMPA full construct.
+
  
−
<br>5/5/15
+
- <br>Transformed ligated plasmid into MG1655-Z1 cells.
  
−
<br>2 colonies from a grown DH5 Z1 plate were selected, 2 cultures inoculated, grown in shaking incubator.
+
- <br>PCR purified INP.  
−
<br>Purify and restriction digest full construct.
+
−
<br>INP digestion.
+
−
<br>Streaked plate with PGSA and BCLA transformed cells.
+
  
−
<br>6/8/15
+
- <br>Did a restriction digestion of INP and Lpp OmpA.
  
−
<br>ZF10 gel electrophoresis.
+
- <br>Ran a gradient PCR of Lpp OmpA full construct.
−
<br>Cut out ZF10 (at 270 bases, according to ladder).
+
−
<br>Gel extraction (freeze ‘n’ squeeze).
+
  
−
<br>7/8/15
+
<br>05/08/2015:
  
−
<br>Streaked chlor plates with transformed RFP DH5 Z1 cells.
+
- <br>2 cultures inoculated with colonies from a DH5α Z1 plate.
−
<br>Re-streaked plates with BCLA and PGSA transformed cells.
+
−
<br>Made chemically competent DH5 Z1 cells (frozen at -80oC).
+
−
vRan gel of digested plasmid, only obtained single band at 2000 base pairs (where backbone without RFP would be, lacking band at 1000 base pairs (for RFP gene)).
+
−
Ran gel of undigested plasmids, single band at 3000 base pairs.
+
−
Gel purified digested plasmid, nanodropped (2.1ng/l).
+
  
 +
- <br>Purified and restriction digested Lpp OmpA full construct.
  
−
<br>8/8/15
+
- <br>Restriction digestion of INP.
  
−
<br>RFP and BCLA transformed cells grow with single colonies.
+
- <br>Streaked plate with pgsA and BclA transformed cells.  
−
<br>PGSA plate showed no growth.
+
−
<br>Inoculated falcon tubes with RFP and BCLA. Also inoculated tube using old PGSA, incubating in shaking incubator.
+
−
Plated pure DH5 Z1 on a chlor and a clean plate (to establish background growth).
+
  
−
<br>9/8/15
+
<br>06/08/2015:
  
−
<br>Miniprep of RFP, BCLA and PGSA transformed cells.
+
- <br>Gel electrophoresis of sZF10.
−
<br>Chlor control plate grew no colonies, clean control plate grew a lawn.  
+
  
 +
- <br>Cut out band corresponding to sZF10 (at 270 bases).
 +
 +
- <br>Did a gel extraction (using Freeze ‘n’ Squeeze method).
 +
 +
<br>07/08/2015:
 +
 +
- <br>Streaked chloramphenicol plates with transformed RFP DH5α Z1 cells.
 +
 +
- <br>Re-streaked plates with BclA and pgsA transformed cells.
 +
 +
- <br>Made chemically competent DH5 α Z1 cells.
 +
 +
- <br>Ran gel of digested plasmid. Obtained single band at 2000 base pairs (where
 +
 +
backbone without RFP would be, lacking band at 1000 base pairs (for RFP gene)).
 +
 +
- <br>Ran gel of undigested plasmids, single band at 3000 base pairs.
 +
 +
- <br>Gel purified digested plasmid, nanodropped (2.1ng/l).
 +
 +
<br>08/08/2015:
 +
 +
- <br>RFP and BclA transformed cells grew with single colonies.
 +
 +
- <br>pgsA plate showed no growth.
 +
 +
- <br>Inoculated falcon tubes with RFP and BclA.
 +
 +
- <br>Inoculated tube using old pgsA.
 +
 +
- <br>Plated pure DH5α Z1 onto 1 chloramphenicol and 1 clean plate (to establish
 +
 +
background growth).
 +
 +
<br>09/08/2015:
 +
 +
- <br>Mini prepped RFP, BclA and pgsA transformed cells.
 +
 +
- <br>Chloramphenicol plate grew no colonies, clean plate grew a lawn.
 
</p>
 
</p>
−
<a href="#0" class="cd-read-more">Read more</a>
+
 
<span class="cd-date">Aug 3</span>
 
<span class="cd-date">Aug 3</span>
 
</div> <!-- cd-timeline-content -->
 
</div> <!-- cd-timeline-content -->
Line 253: Line 300:
 
<p>
 
<p>
  
−
<br>10/8/15
+
<br>10/08/2015:
  
−
<br>Nanodropped miniprep of RFP, BCLA and PGSA.
+
- <br>Nanodropped mini prep of RFP, BclA and pgsA.
−
Sent off for sequencing.  
+
  
−
<br>11/8/15
+
- <br>Sent off for sequencing.
  
−
<br>Digested old EC1 plasmid using Pst1, EcoR1 and Age1, Nde1 to obtain plasmid backbone and full construct … anchor proteins.
+
<br>11/08/2015
−
Digest new RFP plasmid to obtain plasmid backbone.
+
−
Inoculated culture with RFP bacteria, need to mini-prep tomorrow.
+
−
PCR if sZF10.
+
−
Mini-prep of RFP plasmid (JO4450).
+
  
−
<br>12/8/15
+
- <br>Digested old EC1 plasmid using Pst1+EcoR1 and Age1+Nde1 (to obtain
  
−
<br>Prep of 2 binding slides (overnight shaking 37oC, 6 hours 60oC oven, turned halfway) and 1 control (HCL bath only).
+
plasmid backbone, full construct and anchor proteins).
−
Miniprep new RFP plasmid, digest, gelled.
+
−
Gel of old full construct digested by Age1 and Pst1, gel extracted, nanodropped.
+
−
Gel extraction didn’t work.
+
−
Digestion of all zinc fingers.
+
  
−
<br>13/8/15
+
- <br>Digested new RFP plasmid to obtain plasmid backbone.
  
−
<br>Ligation of lpp-full construct into PSB1C3 backbone.
+
- <br>Inoculated culture with RFP bacteria.
−
Transformation of ligation product.
+
  
 +
- <br>PCR of sZF10.
  
 +
- <br>Mini-prep of RFP plasmid (JO4450).
  
−
<br>14/8/15
+
<br>12/08/2015
  
−
<br>Restriction digestion of full construct using Pst1 and EcoR1.
+
- <br>Prepared 2 binding glass slides and 1 control.
−
Inoculation of full construct transformed plasmid.
+
−
Only 2 colonies on the 4 plates of bacteria grew, background growth is 0 via control plates.  
+
  
−
<br>15/8/15
+
- <br>Mini prepped new RFP plasmid.
  
−
<br>Mini-prepped grown transformed DH5 Z1, colonies 1 and 2.
+
- <br>Digested new RFP plasmid and ran it on a gel.  
−
Plated normal DH5 Z1 on clean plates, found lawn, viable cells.
+
−
Re-transformed cells formed a lawn, taken to be re-spread, will inoculate tomorrow.
+
  
 +
- <br>Gel of old full construct digested by Age1+Pst1.
 +
 +
- <br>Gel extracted then nanodropped. Showed that the gel extraction didn’t work.
 +
 +
- <br>Digestion of all zinc fingers.
 +
 +
<br>13/08/2015
 +
 +
- <br>Ligation of Lpp OmpA full construct into pSB1C3backbone.
 +
 +
- <br>Transformation of ligation product.
 +
 +
<br>14/08/2015
 +
 +
- <br>Restriction digestion of Lpp OmpA full construct using Pst1+EcoR1.
 +
 +
- <br>Inoculation of Lpp OmpA full construct transformed plasmid.
 +
 +
- <br>Only 2 colonies on the 4 plates of bacteria grew (background growth is 0 via
 +
 +
control plates).
 +
 +
<br>15/08/2015
 +
 +
- <br>Mini-prepped grown transformed DH5α Z1, colonies 1 and 2.
 +
 +
- <br>Plated normal DH5α Z1 on clean plates (found a lawn of viable cells).
 +
 +
- <br>Re-transformed cells formed a lawn (taken to be re-spread and inoculated).
  
  
 
</p>
 
</p>
−
<a href="#0" class="cd-read-more">Read more</a>
+
 
<span class="cd-date">Aug 10</span>
 
<span class="cd-date">Aug 10</span>
 
</div> <!-- cd-timeline-content -->
 
</div> <!-- cd-timeline-content -->
Line 310: Line 374:
 
<div class="cd-timeline-content">
 
<div class="cd-timeline-content">
 
<h2>Week 8</h2>
 
<h2>Week 8</h2>
−
<p>Lorem ipsum dolor sit amet, consectetur adipisicing elit. Iusto, optio, dolorum provident rerum aut hic quasi placeat iure tempora laudantium ipsa ad debitis unde?</p>
+
<p>
−
<a href="#0" class="cd-read-more">Read more</a>
+
 
 +
<br>17/08/2015:
 +
 
 +
- <br>Prepared mini prep for sequencing.
 +
 
 +
- <br>Digested Lpp OmpA out of the plasmid and gel extracted the backbone.
 +
 
 +
- <br>Ligated all anchor proteins into the plasmid.
 +
 
 +
<br>18/08/2015:
 +
 
 +
- <br>Transformed DH5α Z1 cells with each anchor protein.
 +
 
 +
- <br>Re-growing Lpp OmpA transformed DH5α Z1 cells.
 +
 
 +
- <br>Electroporated MG1655Z1 cells using the original full construct plasmid and
 +
 
 +
plated.
 +
 
 +
- <br>Streaked FIM(negative) cells on a clean plate.
 +
 
 +
<br>19/08/2015:
 +
 
 +
- <br>All plated grew well (Lpp OmpA transformed cells formed a lawn).
 +
 
 +
- <br>Streaked Lpp OmpA transformed cells onto a chloramphenicol plate.
 +
 
 +
- <br>Electroporated cells did not grow.
 +
 
 +
- <br>Inoculations were made of the BclA, pgsA and INP plates.
 +
 
 +
- <br>Made 12 new chloramphenicol plates.
 +
 
 +
- <br>Designed primers (for the DNA origami oligonucleotide adhesive) for the
 +
 
 +
modellers.
 +
 
 +
- <br>Sequences came back clean (with no mutations).
 +
 
 +
- <br>Colony PCR of all grown cells.
 +
 
 +
<br>20/08/2015:
 +
 
 +
- <br>Ran an electrophoresis gel of the colony PCRs.
 +
 
 +
- <br>Made chemically competent FIM(negative).
 +
 
 +
- <br>Made glycerol stocks of all completed cell cultures.
 +
 
 +
- <br>Mini prep of pgsA, BclA and INP plasmids.
 +
 
 +
- <br>Made inoculations of Lpp OmpA (one with IPTG, one without, and one where
 +
 
 +
IPTG is added later).
 +
 
 +
<br>21/08/2015:
 +
 
 +
- <br>BclA, INP and pgsA plasmids sent for sequencing.
 +
 
 +
- <br>Finished making FIM(negative) chemically competent cells.
 +
 
 +
- <br>Measured OD600 of Lpp OmpA inoculations.
 +
 
 +
- <br>Made another Lpp OmpA inoculation.
 +
 
 +
- <br>Remade the colony PCR.
 +
 
 +
<br>22/08/2015:
 +
 
 +
- <br>Ran electrophoresis gel of colony PCR.
 +
 
 +
- <br>No results so colony PCR remade.
 +
 
 +
<br>23/08/2015
 +
 
 +
- <br>No results.
 +
 
 +
- <br>Made inoculations of Lpp OmpA transformed cells (with IPTG and normal DH5α
 +
 
 +
Z1 cells).
 +
</p>
 +
 
<span class="cd-date">Aug 17</span>
 
<span class="cd-date">Aug 17</span>
 
</div> <!-- cd-timeline-content -->
 
</div> <!-- cd-timeline-content -->
Line 327: Line 472:
 
<div class="cd-timeline-content">
 
<div class="cd-timeline-content">
 
<h2>Week 9</h2>
 
<h2>Week 9</h2>
−
<p>Lorem ipsum dolor sit amet, consectetur adipisicing elit. Iusto, optio, dolorum provident rerum aut hic quasi placeat iure tempora laudantium ipsa ad debitis unde?</p>
+
<p>
−
<a href="#0" class="cd-read-more">Read more</a>
+
 
 +
 
 +
<br>24/08/2015:
 +
 
 +
- <br>Made colony PCR again using different primer-colony combinations.
 +
 
 +
- <br>Made more chloramphenicol plates.
 +
 
 +
- <br>Assembled primers.
 +
 
 +
- <br>Made designs (using paper stencils) of RFP transformed DH5α Z1.
 +
 
 +
- <br>Carried out Age1+Nde1 digestion of Lpp OmpA full construct.
 +
 
 +
- <br>Ran electrophoresis gel of colony PCR – had negative results.
 +
 
 +
<br>25/08/2015:
 +
 
 +
- <br>Gel extraction of digested full construct plasmid showed no positive results.
 +
 
 +
- <br>Mini prepped functional cell.
 +
 
 +
- <br>Set up (8 hour) digestion.
 +
 
 +
<br>26/08/2015:
 +
 
 +
- <br>Analyse digestion. Showed a single band (a negative result).
 +
 
 +
- <br>Put DH5αcells on to grow (for microscopy).
 +
 
 +
<br>27/08/2015:
 +
 
 +
- <br>First steps of slide preparation.
 +
 
 +
- <br>Mini prepped Lpp OmpA full construct.
 +
 
 +
- <br>Digested Lpp OmpA full construct with Age1.
 +
 
 +
- <br>Ran an electrophoresis gel of the digestion - results were negative.
 +
 
 +
<br>28/08/2015:
 +
 
 +
- <br>Mini prepped Lpp OmpA full construct.
 +
 
 +
- <br>Ran electrophoresis gel (of digestion with EcoR1+Nde1 and digestion with
 +
 
 +
EcoR1+Age1).
 +
 
 +
- <br>Rerunning electrophoresis gel (using old enzymes and Lpp OmpA plasmid full
 +
 
 +
construct mini prep).
 +
 
 +
<br>29/08/2015:
 +
 
 +
- <br>Did gel extraction of Lpp OmpA full construct plasmid mini prep.
 +
 
 +
- <br>No supernatant after freeze and squeeze step.
 +
 
 +
- <br>MG1655Z1 cells transformed with PSB3K3 via electroporation.
 +
 
 +
<br>30/08/2015:
 +
 
 +
- <br>Redid gel extraction of Lpp OmpA full construct (plasmid mini prep) via.
 +
 
 +
 
 +
 
 +
 
 +
 
 +
</p>
 +
 
<span class="cd-date">Aug 24</span>
 
<span class="cd-date">Aug 24</span>
 
</div> <!-- cd-timeline-content -->
 
</div> <!-- cd-timeline-content -->
Line 340: Line 554:
 
<div class="cd-timeline-content">
 
<div class="cd-timeline-content">
 
<h2>Week 10</h2>
 
<h2>Week 10</h2>
−
<p>Lorem ipsum dolor sit amet, consectetur adipisicing elit. Excepturi, obcaecati, quisquam id molestias eaque asperiores voluptatibus cupiditate error assumenda delectus odit similique earum voluptatem doloremque dolorem ipsam quae rerum quis. Odit, itaque, deserunt corporis vero ipsum nisi eius odio natus ullam provident pariatur temporibus quia eos repellat consequuntur perferendis enim amet quae quasi repudiandae sed quod veniam dolore possimus rem voluptatum eveniet eligendi quis fugiat aliquam sunt similique aut adipisci.</p>
+
<p>
−
<a href="#0" class="cd-read-more">Read more</a>
+
 
 +
 
 +
 
 +
<br>31/08/2015:
 +
 
 +
- <br>Repeated gel extraction (not enough full construct mini prepped plasmid).
 +
 
 +
- <br>Started mini prep of Lpp OmpA full construct transformed colony.
 +
 
 +
- <br>Slide prep up to (but not including) Blocking Buffer wash.
 +
 
 +
- <br>LB inoculated with colony from MG1655Z1 + pSB3K3 plate.
 +
 
 +
<br>01/09/2015
 +
 
 +
- <br>Gel extracted anchor protein plasmids (from digestion).
 +
 
 +
- <br>Microscopy session - some controls not the best, but significant difference in
 +
 
 +
brightness of induced and Lpp OmpA full construct.  
 +
 
 +
<br>02/09/2015:
 +
 
 +
- <br>PCR of sZFs 2, sZF10 sZFand 14.
 +
 
 +
- <br>Mini prepped more plasmid from the working MG1655Z1cells.
 +
 
 +
- <br>Set up more MG1655Z1cells to grow overnight.
 +
 
 +
- <br>Restriction digestion of full construct plasmid carried out using Nhe1+Cla1.
 +
 
 +
<br>03/09/2015:
 +
 
 +
- <br>Triplicate mini prep made using colony 2 (MG1655Z1).
 +
 
 +
- <br>Restriction digested mini prep.
 +
 
 +
- <br>Ran a gel extraction of digestion, then extracted from gel.  
 +
 
 +
- <br>Ran a PCR of the cut zinc fingers.
 +
 
 +
<br>04/09/2015:
 +
 
 +
- <br>Mini prepped a large batch of Dh5α Z1cell.
 +
 
 +
- <br>Ran a restriction digestion (using Age1+Nde1).
 +
 
 +
- <br>JS006 transformed with PSB3K3 via electroporation, grown on 1:2000
 +
 
 +
Kanamycin plate.
 +
 
 +
- <br>Successful PCR of sZF2 and sZF14 (purified and tested).
 +
 
 +
- <br>Redid PCR of sZF10.
 +
 
 +
- <br>Gel extraction of plasmid Mhe1+Cla1 digest, worked perfectly.  
 +
 
 +
- <br>PCR purified the 3 anchor proteins (BclA, pgsA and INP).
 +
 
 +
- <br>Mini prepped anchor proteins.
 +
 
 +
- <br>Digested the anchor proteins, ran on an electrophoresis gel and then extracted.
 +
 
 +
- <br>Carried out a restriction digestion (with Age1+Nde1).
 +
 
 +
- <br>Ran digestion on an electrophoresis gel, and then extracted.
 +
 
 +
<br>05/09/2015
 +
 
 +
- <br>Gel extracted the plasmid digested by Nhe1+Cla1.
 +
 
 +
- <br>Mini prepped more plasmid.
 +
 
 +
- <br>Digestion of plasmid (using Nde1+Age1).
 +
 
 +
- <br>Ran electrophoresis gel of Nde1+Age1 digestion.
 +
 
 +
 
 +
</p>
 +
 
<span class="cd-date">Aug 31</span>
 
<span class="cd-date">Aug 31</span>
 
</div> <!-- cd-timeline-content -->
 
</div> <!-- cd-timeline-content -->
Line 353: Line 646:
 
<div class="cd-timeline-content">
 
<div class="cd-timeline-content">
 
<h2>Week 11</h2>
 
<h2>Week 11</h2>
−
<p>Lorem ipsum dolor sit amet, consectetur adipisicing elit. Iusto, optio, dolorum provident rerum aut hic quasi placeat iure tempora laudantium ipsa ad debitis unde? Iste voluptatibus minus veritatis qui ut.</p>
+
<p>
−
<a href="#0" class="cd-read-more">Read more</a>
+
 
 +
 
 +
<br>07/09/2015:
 +
 
 +
- <br>Ran an electrophoresis gel of Nde1+Age1 digest (reran to separate out bands).
 +
 
 +
- <br>Gel extracted.
 +
 
 +
- <br>Digested all zinc fingers.
 +
 
 +
- <br>Gel of full construct (digested with Nhe1+Cla1) showed a positive result, so PCR
 +
 
 +
purified.
 +
 
 +
- <br>PCR purified sZF2, sZF10 and sZF14.
 +
 
 +
- <br>Ligated (using T4 DNA ligase protocol).
 +
 
 +
<br>08/09/2015:
 +
 
 +
- <br>Ran gel electrophoresis of ligated zinc fingers (to check them).
 +
 
 +
- <br>Transformed some of the ligation into chemically competent DH5α Z1 cells.
 +
 
 +
- <br>Transformed rest of the ligation into electrocompetent cells.
 +
 
 +
- <br>Made more chloramphenicol plates.
 +
 
 +
<br>09/09/2015:
 +
 
 +
- <br>Primer assembly of fluorescent zinc finger specific binding sequences.
 +
 
 +
- <br>Made oligonucleotides for glass DNA binding.
 +
 
 +
- <br>Made fluorescent oligos for specific DNA binding test.
 +
 
 +
<br>10/09/2015:
 +
 
 +
- <br>Plasmid preparation of pSB3K3 for insertion of fluorescent gBlocks.
 +
 
 +
- <br>Wells 6F/18A/18C/18E/18E from kit Plate 4 2015 Phusion PCR.
 +
 
 +
- <br>Gel electrophoresis of PCR products - showed only primer dimer at 100 bp.
 +
 
 +
<br>11/09/2015:
 +
 
 +
- <br>Plasmid preparation of pSB3K3 for insertion of fluorescent gBlocks.
 +
 
 +
- <br>Wells 6F/18A/18C/18E/18E from kit Plate 4 2015 Phusion PCR.
 +
 
 +
- <br>Gel electrophoresis of PCR products - showed only primer dimer at 100 base
 +
 
 +
pairs again.
 +
 
 +
- <br>Mini prepped electroporated MG1655Z1 containing sZFf2, sZF10, sZF14, BclA
 +
 
 +
and INP plasmids.
 +
 
 +
- <br>Sent the 5 modified plasmids for sequencing.
 +
 
 +
- <br>Received PCR product from Oxford iGEM. Assembled oligos in PCR machine.  
 +
 
 +
- <br>Prepared slides (of induced and uninduced zinc fingers) for microscopy using
 +
 
 +
Dh5α Z1 cells.
 +
 
 +
<br>12/09/2015:
 +
 
 +
- <br>Made inoculations of MG1655Z1 transformed cells for preparation of slides
 +
 
 +
(using induced and uninduced zinc fingers and anchors).
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
</p>
 +
 
<span class="cd-date">Sep 07</span>
 
<span class="cd-date">Sep 07</span>
 
</div> <!-- cd-timeline-content -->
 
</div> <!-- cd-timeline-content -->
Line 362: Line 733:
 
<div class="cd-timeline-img cd-location">
 
<div class="cd-timeline-img cd-location">
 
<img src="http://colouringbook.org/SVG/2011/COLOURINGBOOK.ORG/ducky_icon_black_white_line_art_scalable_vector_graphics_svg_inkscape_adobe_illustrator_clip_art_clipart_coloring_book_colouring-1969px.png" alt="Location">
 
<img src="http://colouringbook.org/SVG/2011/COLOURINGBOOK.ORG/ducky_icon_black_white_line_art_scalable_vector_graphics_svg_inkscape_adobe_illustrator_clip_art_clipart_coloring_book_colouring-1969px.png" alt="Location">
−
</div> <!-- cd-timeline-img -->
+
</div> <!-- cd-timeline-img -->
  
 
<div class="cd-timeline-content">
 
<div class="cd-timeline-content">
−
<h2>Week 12</h2>
+
<h2>Final Week</h2>
−
<p>Lorem ipsum dolor sit amet, consectetur adipisicing elit. Iusto, optio, dolorum provident rerum.</p>
+
<p>
−
<a href="#0" class="cd-read-more">Read more</a>
+
−
<span class="cd-date">Sep 14</span>
+
−
</div> <!-- cd-timeline-content -->
+
−
</div> <!-- cd-timeline-block -->
+
  
−
<div class="cd-timeline-block">
 
−
<div class="cd-timeline-img cd-movie">
 
−
<img src="http://colouringbook.org/SVG/2011/COLOURINGBOOK.ORG/ducky_icon_black_white_line_art_scalable_vector_graphics_svg_inkscape_adobe_illustrator_clip_art_clipart_coloring_book_colouring-1969px.png" alt="Movie">
 
−
</div> <!-- cd-timeline-img -->
 
  
−
<div class="cd-timeline-content">
+
<br>14/09/2015
−
<h2>Final Week</h2>
+
 
−
<p>This is the content of the last section</p>
+
- <br>Redo Phusion PCR (gradient) of wells 6F/18A/18C/18E/18E from kit Plate 4
−
<span class="cd-date">Sep 21</span>
+
 
 +
2015 with +/- DMSO.
 +
 
 +
- <br>Gel electrophoresis of PCR products identified band of 2.5kb.
 +
 
 +
- <br>Dpn1 digest for 3 hours and 37°C
 +
 
 +
- <br>Remade glass slides with all zinc fingers (for the oligo binding experiment –
 +
 
 +
experiment 3) and anchor proteins (for FLAG tag experiment – experiment 2).
 +
 
 +
<br>15/09/2015
 +
 
 +
- <br>PCR purification of 18C and 18E.
 +
 
 +
- <br>Gibson assembly of 18C and 18E, each with the gBlock for mKate, GFP and
 +
 
 +
Cerulean
 +
 
 +
- <br>Remade slides with zinc fingers (for experiment 3) and anchor proteins
 +
 
 +
(experiment 2). Found out later that sequencing did not come back right, so slides
 +
 
 +
must be remade using new cells. .
 +
 
 +
<br>16/09/2015
 +
 
 +
- <br>Grew up all zinc fingers cells in m9 (to be used for experiment 2). 
 +
 
 +
<br>17/09/2015
 +
 
 +
- <br>Cells had not grown up, so regrew them in LB.
 +
 
 +
- <br>Once grown, these cells were used to prepare slides for experiment 2.
 +
 
 +
<br> 18/09/2015
 +
 
 +
<br> Visualised slides of zinc finger transformed cells, but only got negative results.
 +
 
 +
<br> Remade slides using alternative protocol (did not fix cells to slide, cells were in liquid media). Visualised, got positive results for sZF14.
 +
 
 +
 
 +
 
 +
 
 +
 
 +
</p>
 +
<span class="cd-date">Sep 14</span>
 
</div> <!-- cd-timeline-content -->
 
</div> <!-- cd-timeline-content -->
 
</div> <!-- cd-timeline-block -->
 
</div> <!-- cd-timeline-block -->
Line 386: Line 795:
 
<script src="http://ajax.googleapis.com/ajax/libs/jquery/1.11.0/jquery.min.js"></script>
 
<script src="http://ajax.googleapis.com/ajax/libs/jquery/1.11.0/jquery.min.js"></script>
 
<script src="js/main.js"></script> <!-- Resource jQuery -->
 
<script src="js/main.js"></script> <!-- Resource jQuery -->
 +
 +
 
</body>
 
</body>
  
  
  
−
<b></b><a href="#menu-toggle" class="btn btn-default" id="menu-toggle">Toggle Menu</a></p>
+
−
<script>$("#menu-toggle").click(function(e) {
+
 
−
        e.preventDefault();
+
−
        $("#wrapper").toggleClass("toggled");
+
−
    });</script>
+
  
 
</html>
 
</html>

Latest revision as of 20:11, 18 September 2015

Warwick iGEM 2015

Open Menu