Team:Aachen/Notebook/Wetlab/May

From 2014.igem.org

(Difference between revisions)
(21th)
 
(6 intermediate revisions not shown)
Line 8: Line 8:
<ul class="menusmall-grid">
<ul class="menusmall-grid">
-
  <li style="width:106px;margin-left: 12px;margin-right: 12px;" >
+
<!--  <li style="width:106px;margin-left: 12px;margin-right: 12px;" >
     <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/March" style="color:black">
     <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/March" style="color:black">
     <div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel" style="top: 30%; font-size: 16px;">Go to March</div></div>
     <div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel" style="top: 30%; font-size: 16px;">Go to March</div></div>
Line 14: Line 14:
     </div>
     </div>
     </a>
     </a>
-
   </li>
+
   </li> -->
   <li style="width:106px;margin-left: 12px;margin-right: 12px;" >
   <li style="width:106px;margin-left: 12px;margin-right: 12px;" >
Line 88: Line 88:
== 8th ==
== 8th ==
* The efficiency of our competent cells was tested
* The efficiency of our competent cells was tested
-
*: &rarr; BL21: 6.6 x 10<sup>4</sup>
+
*: &rarr; BL21: 6.6 x 10<sup>4</sup> clones/µg DNA
-
*: &rarr; DH5α: 2.59 x 10<sup>7</sup>
+
*: &rarr; DH5α: 2.59 x 10<sup>7</sup> clones/µg DNA
* SOE-PCR step 2 like on 30.04. with the template of SOE1 from 30.04. was re-done
* SOE-PCR step 2 like on 30.04. with the template of SOE1 from 30.04. was re-done
* The SOE2 product was run on a gel for checking (5&nbsp;µL)
* The SOE2 product was run on a gel for checking (5&nbsp;µL)
Line 97: Line 97:
== 14th ==
== 14th ==
* LB agar plates with chloramphenicol and some with ampicillin were made
* LB agar plates with chloramphenicol and some with ampicillin were made
-
* REACh2 was purified on 1.2&nbsp;% agarose gel
+
* REACh2 was purified on 1.2% agarose gel
* A subsequent purification of the 778 bp fragment with High Pure PCR Product Purification Kit was done
* A subsequent purification of the 778 bp fragment with High Pure PCR Product Purification Kit was done
== 19th ==
== 19th ==
-
* K131026 was transformed into DH5α and NEB
+
* K131026 (AHL inducible GFP) was transformed into DH5α and NEB
-
* K731520 was transformed into DH5α
+
* K731520 (IPTG inducible GFPmut3b) was transformed into DH5α
== 20th ==
== 20th ==
-
* Master plates on chloramphenicol (cam) &rarr; at least 6 clones on each plate
+
* Master plates with chloramphenicol (cam) &rarr; at least 6 clones on each plate
* 2x 5&nbsp;mL LB + cam were prepared
* 2x 5&nbsp;mL LB + cam were prepared
-
* Sterile 50&nbsp;% glycerol was made
+
* Sterile 50% glycerol was made
== 21th ==
== 21th ==
* Cryo stocks of clones 1 & 2 of each BioBrick/ host were prepared
* Cryo stocks of clones 1 & 2 of each BioBrick/ host were prepared
* 15&nbsp;mL cultures in 250&nbsp;mL flasks, inoculated with 1.5&nbsp;mL preculture (3 cultures A B C from clones 1; 1 + 2; 2)
* 15&nbsp;mL cultures in 250&nbsp;mL flasks, inoculated with 1.5&nbsp;mL preculture (3 cultures A B C from clones 1; 1 + 2; 2)
-
* 250&nbsp;µl Chloramphilicol from a 35&nbsp;mg/mL stock was added
+
* 250&nbsp;µl cam from a 35&nbsp;mg/mL stock was added
* The cultures were grown until OD<sub>600</sub>= 0.6, then one was induced with IPTG
* The cultures were grown until OD<sub>600</sub>= 0.6, then one was induced with IPTG
<center>
<center>
Line 141: Line 141:
 +
<html>
<center>
<center>
-
{| class="wikitable"
+
<ul class="menusmall-grid">
-
|align="left"| [[Team:Aachen/Notebook/Wetlab/April|previous month]] || align="center"| [[Team:Aachen/Notebook/Wetlab2|Wetlab main page]] || align="right"| [[Team:Aachen/Notebook/Wetlab/June|next month]]
+
-
|}
+
-
</center>
+
 +
<!--  <li style="width:106px;margin-left: 12px;margin-right: 12px;" >
 +
    <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/March" style="color:black">
 +
    <div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel" style="top: 30%; font-size: 16px;">Go to March</div></div>
 +
    <div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/7/7a/Aachen_14-10-10_March_iFG.png); norepeat scroll 0% 0% transparent; background-size:100%; height:100px; width: 100px;">
 +
    </div>
 +
    </a>
 +
  </li> -->
 +
 +
  <li style="width:106px;margin-left: 12px;margin-right: 12px;" >
 +
    <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/April" style="color:black">
 +
    <div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel" style="top: 30%; font-size: 16px;">Go to April</div></div>
 +
    <div class="menusmall-item menusmall-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%; height:100px; width: 100px;">
 +
    </div>
 +
    </a>
 +
  </li>
 +
 +
  <li style="width:106px;margin-left: 12px;margin-right: 12px;" >
 +
    <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/May" style="color:black">
 +
    <div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel" style="top: 30%; font-size: 16px;">Go to May</div></div>
 +
    <div class="menusmall-item menusmall-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%; height:100px; width: 100px;">
 +
    </div>
 +
    </a>
 +
  </li>
 +
 +
  <li style="width:106px;margin-left: 12px;margin-right: 12px;" >
 +
    <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/June" style="color:black">
 +
    <div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel" style="top: 30%; font-size: 16px;">Go to June</div></div>
 +
    <div class="menusmall-item menusmall-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%; height:100px; width: 100px;">
 +
    </div>
 +
    </a>
 +
  </li>
 +
 +
  <li style="width:106px;margin-left: 12px;margin-right: 12px;" >
 +
    <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/July" style="color:black">
 +
    <div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel" style="top: 30%; font-size: 16px;">Go to July</div></div>
 +
    <div class="menusmall-item menusmall-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%; height:100px; width: 100px;">
 +
    </div>
 +
    </a>
 +
  </li>
 +
 +
  <li style="width:106px;margin-left: 12px;margin-right: 12px;" >
 +
    <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/August" style="color:black">
 +
    <div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel" style="top: 30%; font-size: 16px;">Go to August</div></div>
 +
    <div class="menusmall-item menusmall-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%; height:100px; width: 100px;">
 +
    </div>
 +
    </a>
 +
  </li>
 +
 +
  <li style="width:106px;margin-left: 12px;margin-right: 12px;" >
 +
    <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/September" style="color:black">
 +
    <div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel" style="top: 30%; font-size: 16px;">Go to September</div></div>
 +
    <div class="menusmall-item menusmall-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%; height:100px; width: 100px;">
 +
    </div>
 +
    </a>
 +
  </li>
 +
 +
  <li style="width:106px;margin-left: 12px;margin-right: 12px;" >
 +
    <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/October" style="color:black">
 +
    <div class="menusmall-item menusmall-info" style="height:100px; width: 100px;" ><div class="menukachel" style="top: 30%; font-size: 16px;">Go to October</div></div>
 +
    <div class="menusmall-item menusmall-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%; height:100px; width: 100px;">
 +
    </div>
 +
    </a>
 +
  </li>
 +
 +
</ul>
 +
</center>
 +
</html>
{{Team:Aachen/Footer}}
{{Team:Aachen/Footer}}

Latest revision as of 16:11, 17 October 2014

May

1st

  • A gel with M - full -full - REACh1 SOE3.2 - REACH2 SOE3.2 - M was run
    → 120 V, 30 min
    → cut out the bands

5th

  • Chemical competent DH5α and BL21 cells were made

8th

  • The efficiency of our competent cells was tested
    → BL21: 6.6 x 104 clones/µg DNA
    → DH5α: 2.59 x 107 clones/µg DNA
  • SOE-PCR step 2 like on 30.04. with the template of SOE1 from 30.04. was re-done
  • The SOE2 product was run on a gel for checking (5 µL)
    → restriction, (dephosphorylation of vector)
    → purification on a gel with high pure kit

14th

  • LB agar plates with chloramphenicol and some with ampicillin were made
  • REACh2 was purified on 1.2% agarose gel
  • A subsequent purification of the 778 bp fragment with High Pure PCR Product Purification Kit was done

19th

  • K131026 (AHL inducible GFP) was transformed into DH5α and NEB
  • K731520 (IPTG inducible GFPmut3b) was transformed into DH5α

20th

  • Master plates with chloramphenicol (cam) → at least 6 clones on each plate
  • 2x 5 mL LB + cam were prepared
  • Sterile 50% glycerol was made

21th

  • Cryo stocks of clones 1 & 2 of each BioBrick/ host were prepared
  • 15 mL cultures in 250 mL flasks, inoculated with 1.5 mL preculture (3 cultures A B C from clones 1; 1 + 2; 2)
  • 250 µl cam from a 35 mg/mL stock was added
  • The cultures were grown until OD600= 0.6, then one was induced with IPTG
time info/ OD
11:10A: inoculated B: inoculated C: inoculated
11:38A: 0.482 B: 0.464 C: 0.466
12:28A: 0.586; induced with 0.1 mM IPTG B: 0.576 C: 0.568
14:11A: 0.93 B: 0.91 C: 0.942

→ A negative control without plasmid was left out

23rd

  • Some BioBricks were transformed
  • Colony PCR (s. picture below)
    → K131026: 1807 bp
    → K731520: 2123 bp
    → full length EYFP: 778 bp
Aachen 14-05-23 COLONY-PCR.jpg
Colony PCR
todo