Team:Aachen/Notebook/Wetlab/May

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<!--  <li style="width:106px;margin-left: 12px;margin-right: 12px;" >
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    <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/March" style="color:black">
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    <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>
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    <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/April" style="color:black">
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    <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>
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    <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;">
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    <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/May" style="color:black">
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    <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>
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    <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;">
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    <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/June" style="color:black">
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  <li style="width:106px;margin-left: 12px;margin-right: 12px;" >
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    <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/July" style="color:black">
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    <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>
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    <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;">
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    <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/August" style="color:black">
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    <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>
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    <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;">
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    <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/September" style="color:black">
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    <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>
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    <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;">
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    <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/October" style="color:black">
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    <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>
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    <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;">
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= May =
= May =
== 1st ==
== 1st ==
-
* gel with M - full -full - REACh1 SOE3.2 - REACH2 SOE3.2 - M
+
* A gel with M - full -full - REACh1 SOE3.2 - REACH2 SOE3.2 - M was run
*: &rarr; 120&nbsp;V, 30 min
*: &rarr; 120&nbsp;V, 30 min
*: &rarr; cut out the bands
*: &rarr; cut out the bands
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== 5th ==
== 5th ==
-
* made chemical competent DH5α and BL21 cells
+
* Chemical competent DH5α and BL21 cells were made
== 8th ==
== 8th ==
-
* 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
-
* 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)
*: &rarr; restriction, (dephosphorylation of vector)
*: &rarr; restriction, (dephosphorylation of vector)
*: &rarr; purification on a gel with high pure kit
*: &rarr; purification on a gel with high pure kit
== 14th ==
== 14th ==
-
* made some LB agar plates with chloramphenicol and some with ampicillin
+
* LB agar plates with chloramphenicol and some with ampicillin were made
-
* REACh2 purification on 1.2&nbsp;% agarose gel
+
* REACh2 was purified on 1.2% agarose gel
-
* subsequent purification of the 778 bp fragment with High Pure PCR Product Purification Kit
+
* A subsequent purification of the 778 bp fragment with High Pure PCR Product Purification Kit was done
== 19th ==
== 19th ==
-
* transformation of K131026 in DH5α and NEB
+
* K131026 (AHL inducible GFP) was transformed into DH5α and NEB
-
* transformation of K731520 in DH5α
+
* K731520 (IPTG inducible GFPmut3b) was transformed into DH5α
== 20th ==
== 20th ==
-
* made 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
-
* prepared 2x 5&nbsp;mL LB + cam
+
* 2x 5&nbsp;mL LB + cam were prepared
-
* made sterile 50&nbsp;% glycerol
+
* 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
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-
&rarr; a negative control without plasmid was left out
+
&rarr; A negative control without plasmid was left out
== 23rd ==
== 23rd ==
-
* transformation of some BioBricks
+
* Some BioBricks were transformed
-
* colony PCR (s. picture below)
+
* Colony PCR (s. picture below)
*: &rarr; K131026: 1807&nbsp;bp
*: &rarr; K131026: 1807&nbsp;bp
*: &rarr; K731520: 2123&nbsp;bp
*: &rarr; K731520: 2123&nbsp;bp
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{| border="0"
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<ul class="menusmall-grid">
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|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]]
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<!--  <li style="width:106px;margin-left: 12px;margin-right: 12px;" >
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    <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/March" style="color:black">
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    <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>
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  <li style="width:106px;margin-left: 12px;margin-right: 12px;" >
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    <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;">
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  <li style="width:106px;margin-left: 12px;margin-right: 12px;" >
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    <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;">
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    <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/June" style="color:black">
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    <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;">
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  <li style="width:106px;margin-left: 12px;margin-right: 12px;" >
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    <a class="menulink" href="https://2014.igem.org/Team:Aachen/Notebook/Wetlab/July" style="color:black">
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    <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;">
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  <li style="width:106px;margin-left: 12px;margin-right: 12px;" >
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    <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;">
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  <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;">
 +
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 +
    <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;">
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{{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