Team:Aachen/Notebook/Wetlab

From 2014.igem.org

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__NOTOC__
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{| cellpadding="10"
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=Our Work in the Lab=
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| __TOC__
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| In order to detect the pathogens fast, specifically and inexpensively we are building sensor cells to detect these pathogens. These sensor cells can identify pathogens in very low concentration by responsing to specific extracellular molecules either secreted by or displayed on the pathogens. These molecules trigger a fast fluorescence response by our immobilized sensor cells  which will be measured by our device.
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| [[File:Aachen_Cellock_standingup.png|300px]]
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|}
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<center>
 +
<html><ul class="team-grid" style="width:1064px;">
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<!-- Overview -->
 +
<li style="width:220px;margin-left: 23px;margin-right: 23px;margin-bottom: 23px;margin-top: 23px;">
 +
<a href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/April" style="color:black">
 +
    <div class="team-item team-info" style="width:214px;height:214px;" >
 +
      <br/><br/>
 +
<b>
 +
preparation of competent cells
 +
<br/><br/>
 +
test on transformation efficiency
 +
<br/><br/>
 +
development of quenchers
 +
</b>
 +
      <br/><br/>
 +
      <!-- click for more information -->
 +
    </div>
 +
    <div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/2/2d/Aachen_14-10-10_April_iFG.png); norepeat scroll 0% 0% transparent; background-size:100%;width:214px;height:214px;"> </div></a>
 +
  </li>
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More information will be available soon!
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<li style="width:220px;margin-left: 23px;margin-right: 23px;margin-bottom: 23px;margin-top: 23px;">
 +
<a href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/May" style="color:black">
 +
    <div class="team-item team-info" style="width:214px;height:214px;" >
 +
      <br/><br/>
 +
<b>
 +
development of quenchers
 +
<br/><br/>
 +
first transformation of BioBricks
 +
<br/><br/>
 +
test of BioBricks
 +
</b>
 +
      <br/><br/>
 +
      <!-- click for more information -->
 +
    </div>
 +
    <div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/6/67/Aachen_14-10-10_May_iFG.png); norepeat scroll 0% 0% transparent; background-size:100%;width:214px;height:214px;"> </div></a>
 +
  </li>
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= May =
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<li style="width:220px;margin-left: 23px;margin-right: 23px;margin-bottom: 23px;margin-top: 23px;">
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== 8th ==
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<a href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/June" style="color:black">
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* SOE-PCR step 2
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    <div class="team-item team-info" style="width:214px;height:214px;" >
 +
      <br/><br/>
 +
<b>
 +
BioBrick transformation marathon
 +
<br/><br/>
 +
construction of first own BioBrick
 +
<br/><br/>
 +
processing of Biobricks
 +
</b>
 +
      <br/><br/>
 +
      <!-- click for more information -->
 +
    </div>
 +
    <div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/1/1d/Aachen_14-10-10_June_iFG.png); norepeat scroll 0% 0% transparent; background-size:100%;width:214px;height:214px;"> </div></a>
 +
  </li>
-
== 23rd ==
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<li style="width:220px;margin-left: 23px;margin-right: 23px;margin-bottom: 23px;margin-top: 23px;">
-
* we transformed some BioBricks and did a colony PCR
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<a href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/July" style="color:black">
-
{{Team:Aachen/Figure|Aachen_14-05-23_COLONY-PCR.jpg|title=Colony PCR picture|subtitle=todo|width=300px}}
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    <div class="team-item team-info" style="width:214px;height:214px;" >
 +
      <br/><br/>
 +
<b>
 +
preparation of 2D-detection chips
 +
<br/><br/>
 +
testing our chip system
 +
<br/><br/>
 +
development of chip measurement methods
 +
</b>
 +
      <br/><br/>
 +
      <!-- click for more information -->
 +
    </div>
 +
    <div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/1/19/Aachen_14-10-10_July_iFG.png); norepeat scroll 0% 0% transparent; background-size:100%;width:214px;height:214px;"> </div></a>
 +
  </li>
 +
</ul></html>
 +
</center>
-
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<center>
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<html><ul class="team-grid" style="width:798px;">
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<!-- Overview -->
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{{Team:Aachen/JumpUp}}
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<li style="width:220px;margin-left: 23px;margin-right: 23px;margin-bottom: 23px;margin-top: 23px;">
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<a href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/August" style="color:black">
-
= June =
+
    <div class="team-item team-info" style="width:214px;height:214px;" >
-
== 25th ==
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      <br/><br/>
-
* preculture for competent NEB10β cells
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<b>
-
* submit samples for sequencing
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start of Gal3 approach
-
* make masterplates of transformed BioBricks
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<br/><br/>
-
* centrifuge and freeze overnight expression culture of J23101.E0240 and K516132
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detection of HSL with chip system
-
 
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<br/><br/>
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== 26th ==
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testing of <i> Pseudomonas fluorescens </i>
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* made competent NEB10β cells - several things went wrong.. we'll see (remember: pre-cool centrifuge, always check if it's indeed spinning, frequently check OD of the culture)
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</b>
-
* colony PCR on the transformed clones looked awful. There were too many cells in the 10&nbsp;µl reaction volume. Some seals weren't fully closed. Basically ''only'' primers and smear except for the positive control which contained a plasmid template instead of cells
+
      <br/><br/>
-
* 2x 500&nbsp;ml of LB and three sterile flasks have been made
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      <!-- click for more information -->
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+
    </div>
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= July =
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    <div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/4/4e/Aachen_14-10-10_August_iFG.png); norepeat scroll 0% 0% transparent; background-size:100%;width:214px;height:214px;"> </div></a>
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== 16th ==
+
  </li>
-
* plasmid preps
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* transformation of different reporter strains
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-
** NEB
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***pSEVE641_BsFbFP
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-
***pSEVA234_LasR
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***pSEVE641_BsFbFP pSEVA234_LasR
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-
***pSEVE641_BsFbFP pSB1C3_C0179
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**BL21
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***pSEVE641_BsFbFP pSEVA234_LasR
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***pSEVE641_BsFbFP pSB1C3_C0179
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-
 
+
-
== 22nd ==
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-
* [[User:Pdemling|Philipp]] and [[User:mosthege|Michael]] made 100x stocks for Hartmans media
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-
* Michael and [[User:VeraA|Vera]] made about 25 HM+C-plates without Glucose
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-
* K731520 iLOV was plated on a HM+C plate
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-
 
+
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== 23rd ==
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* [[User:mosthege|Michael]] made 25 new HM+C plates with 1&nbsp;% agar and 4&nbsp;g/L glucose
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-
* He also added 170&nbsp;µl of the Glucose stock (500&nbsp;g/L) to the Glucose-free HM+C-plates
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* 1&nbsp;L of sterile HM+Glucose was prepared
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-
* K731520 iLOV was plated on HM+C+Glucose and HM+C+Glucose drops, 3x 5&nbsp;ml HM+C+Glucose precultures were inoculated (17:00)
+
-
 
+
-
== 24th ==
+
-
* K731520 iLOV did not grow, neither on the plates, nor on in the liquid media. The most probable cause is that the E.&nbsp;coli is missing some vitamins
+
-
* based upon the [http://openwetware.org/wiki/M9_medium/supplemented M9 recipes by the Knight and Endy labs on OpenWetWare], we made a 20&nbsp;ml supplement stock solution that can be added to 1x HM media
+
-
 
+
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{| class="wikitable"
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|-
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! Component !! supplement for 1x HM [g/L] !! final 1x concentration
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-
|-
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| Casamino acids || 2 || 2 g/L
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-
|-
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| Thiamine hydrochloride || 0.3 || 300 mg/L
+
-
|-
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| MgSO{{sub|4}}/MgSO{{sub|4}}*7H{{sub|2}}O || 0.242 / 0,494 || 2 mmol/L
+
-
|-
+
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| CaCl{{sub|2}} || 0.011 || 0.1 mmol/L
+
-
|}
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-
The powders for 1&nbsp;L of 1x HM were dissolved in 20&nbsp;ml of a 10&nbsp;%&nbsp;w/v Casamino acid stock solution and sterile-filtered (.22&nbsp;µm PES). To make agar chips, we can add 1&nbsp;ml to the hot agar mixture, together with the three 500&nbsp;µl 100x stocks for the HM.
+
-
 
+
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We made 250&nbsp;ml of HM+C+Glucose+Supplements plates with 1.5&nbsp;% agar.
+
-
 
+
-
Then we plated the following combinations:
+
-
 
+
-
{| class="wikitable"
+
-
|-
+
-
! Construct !! HM+C+Glucose+Supplements !! LB+C
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-
|-
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| K731520 iLOV || - || +
+
-
|-
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| J23101.E0240 || + || +
+
-
|}
+
-
 
+
-
We also prepared a 150&nbsp;ml HM+C+Glucose+Supplements culture with K731520 iLOV to hopefully make agar chips on Friday.
+
-
 
+
-
6x of last weeks J23115.E0240 clones were plated on LB+C and 5&nbsp;ml LB+C precultures were inoculated for plasmid preparation.
+
-
 
+
-
== 25th ==
+
-
The K731520 iLOV strain did not grow on the HM plate, while another strain with the J23101.E0240 construct did. Both have the pSB1C3 backbone. To confirm the plasmids and inserts, Michael set up a colony PCR:
+
-
 
+
-
{| class="wikitable"
+
-
|-
+
-
! ID !! Template !! Product Length !! Result
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-
|-
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-
| 1 || J23101.E0240 #5 || 1233 ||
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-
|-
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| 2 || J23101.E0240 #6 || 1233 ||
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|-
+
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| 3 || J23101.E0240 #5 plasmid || 1233 ||
+
-
|-
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-
| 4 || K731520 iLOV LB colony || 2053  ||
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-
|-
+
-
| 5 || K731520 iLOV plasmid || 2053 ||
+
-
|-
+
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| 6 || K731520 GFP plasmid || 2437 ||
+
-
|-
+
-
| 7 || water || none ||
+
-
|}
+
-
 
+
-
Reaction volume per tube is 15&nbsp;µl, GoTaq Green Mastermix and the VF2 and VR primers. You can find the durations and temperatures are in this table:
+
-
 
+
-
{| class="wikitable"
+
-
! parameter !! duration !! temp [°C]
+
-
|-
+
-
| denature||10:00||95
+
-
|-
+
-
| '''denature'''||00:30||95
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-
|-
+
-
| '''anneal'''||00:30||49
+
-
|-
+
-
| '''elongate'''||02:36||72
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-
|-
+
-
| elongate||05:00||72
+
-
|-
+
-
| store||forever||8
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-
|}
+
-
 
+
-
[[User:fgohr|Florian]] mini-prepped the 6 overnight cultures of J23115.E0240 clones. He used 3&nbsp;ml instead of 1.5 and eluted twice with 25&nbsp;µl nuclease-free water. Everything else was according to the protocol of the illustra plasmidPrep Mini-Spin Kit.
+
-
 
+
-
The resulting DNA concentrations are listed in this table:
+
-
 
+
-
{| class="wikitable"
+
-
! clone # !! concentration [ng/µl]
+
-
|-
+
-
| 1 || 73.5
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-
|-
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-
| 2 || 94.5
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-
|-
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| 3 || 100.5
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|-
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| 4 || 53
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|-
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| 5 || 82
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|-
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| 6 || 138
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-
|}
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+
-
In the evening, we plated some strains on the different media to investigate why the K731520 iLOV did not grow on the HM+suppl. plate. (Note: in the morning, Michael re-inoculated the HM+C+suppl. shake flask with cells from the LB-plate and by afternoon the culture ''did'' grow to a high cell density.)
+
-
 
+
-
{| class="wikitable"
+
-
! Construct !! Host strain !! LB+C !! HM+C+Glucose !! HM+C+Glucose+supplements
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-
|-
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| J23101.E0240 || NEB10beta || ++ || - || ++
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-
|-
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-
| K731520 iLOV || DH5alpha || ++ || - || (+)
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-
|-
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-
| K131026 || NEB10beta || ++ || - || -
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|-
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-
| K131026 || DH5alpha || ++ || - || +
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-
|}
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-
 
+
-
Philipp inoculated J23101.E0240 in LB and HM+C+Glucose+supplements
+
-
 
+
-
 
+
-
== 28th ==
+
-
<!-- WICHTIG: Unterlagen für Zollkram heraussuchen und der Frau von der ZHV zurückschreiben!!!! -->
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-
== 29th ==
+
-
 
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* ...
+
-
 
+
-
<!--
+
-
* inoculate 2x 150&nbsp;ml cultures HM+Glucose+C
+
-
* prepare 200&nbsp;ml 1.5&nbsp;% agar
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-
* pre-cool centrifuge
+
-
* centrifuge 1x 50, 1x 100 and 1x 150&nbsp;ml
+
-
* make 3 kinds of chips - OOx1, ODx2, ODx3
+
-
 
+
-
-->
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-
 
+
-
= August =
+
-
== 1st ==
+
-
* Stefan and Vera made electrocompetent ''E.coli''.
+
-
* Arne prepared cultures for Saturday, 14-8-2 of Renés iLOV and K131026
+
-
 
+
-
== 2nd ==
+
-
* Testing the OD measurement device and comparing it to the Spektrophotometer and the Platereader.
+
-
* Also testing K131026 and K731520 iLOV for fluorescence in the Platereader.
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* Heat shock Transformation into NEB TOP 10 cells of I746909.
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* Electroshock transformation of pET17-Gal3 into ''E.coli rosetta''.
+
-
 
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== 3rd ==
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* OD Measurements of the iGEM device in comparison to the Spektrometer.
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* Preparing culutures for cryos of K131026 and K731520 iLOV
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-
 
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== 4th ==
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* Arne and Michael made cryo-stocks of K731520 iLOV and K131026 in NEB/BL21/DH5alpha, I746909 in BL21 and pET17-His-SNAP-YFP-Gal3 in ''E.&nbsp;coli'' Rosetta (DE3)
+
-
* Michael plasmid-prepped most of them using 1.5&nbsp;ml culture and eluted with 1x 50&nbsp;µl of ddH{{sub|2}}O.
+
-
 
+
-
{| class="wikitable"
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-
! combination !! concentration [ng/µl]
+
-
|-
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| I746909 BL21 #1 || 73.5
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-
|-
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-
| I746909 BL21 #2 || 45
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-
|-
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| I746909 BL21 #3 || 49
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-
|-
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| K731520 iLOV DH5alpha || 60
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-
|-
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| K131026 DH5alpha || 150
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-
|-
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| pET17-Gal3 #1 || 30.5
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-
|-
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| pET17-Gal3 #2 || 6.4
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-
|-
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| pET17-Gal3 #3 || 6.3
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-
|-
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| pET17-Gal3 #4 || 9.4
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-
|-
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| pET17-Gal3 #5 || 10.1
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-
|-
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| pET17-Gal3 #6 || 8.2
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-
|-
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| pET17-Gal3 #7 || 13.8
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-
|-
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| pET17-Gal3 #8 || 6.9
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-
|-
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| pET17-Gal3 #9 || 10.2
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-
|}
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-
 
+
-
To confirm the quality of pET17-Gal3-transformations, the purified plasmids were test-digested:
+
-
 
+
-
{{Team:Aachen/Figure|14-08-04_Test-Digest.png|title=Test digest|subtitle=clones were test-digested|width=200px}}
+
-
 
+
-
{| class="wikitable"
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-
! combination !! cut products[bp]
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-
|-
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| I746909 BL21 #1 || 2029, 947
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-
|-
+
-
| K731520 iLOV DH5alpha || 2029, 1780
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-
|-
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-
| K131026 DH5alpha || 2029, 1848
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-
|-
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-
| pET17-Gal3 #1 || 3086, 923, 1262
+
-
|}
+
-
 
+
-
All pET17-Gal3 clones were positive and clone #2 was selected for further experiments.
+
-
 
+
-
== 5th ==
+
-
* Stefan, Arne and Michael assembled a V{{sub|R}}=2.5&nbsp;L bioreactor for cultivation of a 1&nbsp;L expression culture
+
-
* Two precultures of 20&nbsp;ml LB+A were inoculated at 19:00
+
-
* Anna transformed K746909 into BL21 cells and K1319000 into NEB10beta
+
-
 
+
-
== 6th ==
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-
* Eshani and Arne transformed J04450 in pSB1K3 and pSB1A3 in NEB Beta cells
+
-
* They also did a plasmid prep of J04450 in pSB1C3 and Flo's vectors
+
-
* Arne made precultures of NEB Beta and DH5 alpha cells
+
-
* Arne and Michael inoculated the fermenter at 11:40, induced the fermentation of pET17-Gal3. The fermentation is expected to run 24&nbsp;h
+
-
 
+
-
== 25th ==
+
-
* Plasmid restriction of I20260 (EcoRI,PstI), J23115 (EcoRI, SpeI), K516032 (XbaI,PstI), and J23101 (EcoRI, SpeI)
+
-
 
+
-
== 26th ==
+
-
* Ligation of J23115 and K516032 to J23115.K516032 and J23101 and K516032 to J23101.K516032
+
-
* Plasmid prep of I20260, K516032 and B0034
+
-
* Restriction of Plasmid I20260, K516032, B0034 with EcoRI and PstI
+
-
* Gel with restricted I20260, K516032 and B0034
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-
* purification of vector backbone, pSB1A2, pSB3K3 and pSB1C3
+
-
* Restriciton of snythesized TEV-Protease with EcoRI and PstI
+
-
 
+
-
== 27th ==
+
-
* Ligation of J23101.K516032 into pSB3K3 and J23115.K516032 into pSB3K3 and K1319004 into pSB1C3
+
-
* Transformation of K1319004 in pUC and pSB1C3 as well as J04450 in pSB1K3 and pSB1A3
+
-
 
+
-
= September =
+
-
== 3rd ==
+
-
Michael, Vera and Eshani prepared 50&nbsp;ml LB+antibiotic overnight-cultures of pSBX-vectors from Heidelberg.
+
-
 
+
-
== 04th ==
+
-
Michael and Anna did PCRs for Gibson-Assembly of K1319003 into pET17. Duplicates of 25&nbsp;µl reaction volume (12.5&nbsp;µl Q5 2x Master Mix, 2x 1.25&nbsp;µl primers, 2&nbsp;µl template)
+
-
 
+
-
{| class="wikitable"
+
-
! PCR tube # !! components
+
-
|-
+
-
| 1 and 2 || pET17 + pET17_Gal3_Gib_F + pET17_Gal3_Gib_R
+
-
|-
+
-
| 3 and 4 || K1319003 + K1319003_Gib_F + K1319003_Gib_R
+
-
|-
+
-
|}
+
-
 
+
-
The PCR conditions:
+
-
 
+
-
{| class="wikitable"
+
-
! step !! temperature [°C] !! duration
+
-
|-
+
-
| denature || 98 || 30", 98 °C for 10", 55 °C for 30", 72 °C for 2'15"
+
-
|-
+
-
| denature || 98 || 10"
+
-
|-
+
-
| anneal || 50 (insert) 55 (backbone) || 30"
+
-
|-
+
-
| elongate || 72 || 0'30" (insert) 2'15" (backbone)
+
-
|-
+
-
| elongate || 72 || 2"
+
-
|-
+
-
| store || 8 || indefinite
+
-
|}
+
-
 
+
-
== 05th ==
+
-
 
+
-
= October =
+
-
...
+
 +
<li style="width:220px;margin-left: 23px;margin-right: 23px;margin-bottom: 23px;margin-top: 23px;">
 +
<a href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/September" style="color:black">
 +
    <div class="team-item team-info" style="width:214px;height:214px;" >
 +
      <br/><br/>
 +
<b>
 +
interlab study
 +
<br/><br/>
 +
construction of GFP-quencher-fusions
 +
<br/><br/>
 +
further devolopment of BioBricks
 +
</b>
 +
      <br/><br/>
 +
      <!-- click for more information -->
 +
    </div>
 +
    <div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/d/d4/Aachen_14-10-10_September_iFG.png); norepeat scroll 0% 0% transparent; background-size:100%;width:214px;height:214px;"> </div></a>
 +
  </li>
 +
<li style="width:220px;margin-left: 23px;margin-right: 23px;margin-bottom: 23px;margin-top: 23px;">
 +
<a href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/October" style="color:black">
 +
    <div class="team-item team-info" style="width:214px;height:214px;" >
 +
      <br/><br/>
 +
<b>
 +
working 24/7
 +
<br/><br/>
 +
finalizing our project
 +
<br/><br/>
 +
Wiki
 +
</b>
 +
      <br/><br/>
 +
      <!-- click for more information -->
 +
    </div>
 +
    <div class="team-item team-img" style="background: url(https://static.igem.org/mediawiki/2014/6/60/Aachen_14-10-10_October_iFG.png); norepeat scroll 0% 0% transparent; background-size:100%;width:214px;height:214px;"> </div></a>
 +
  </li>
 +
</ul></html>
 +
</center>
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Latest revision as of 21:23, 17 October 2014

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