Team:Goettingen/week5-2

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<li>Experiment:  <br>
<li>Experiment:  <br>
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In order to clone <i>flhDC</i> behind the different promoters, the purified PCR product was digested with <i>XbaI</i> and <i>PstI</i>, whereas the BioBrick plasmids were cut with <i>SpeI</i> and </i> PstI. The digestion was performed as described in the <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a>. At first only three promoters were chosen: <br>
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In order to clone <i>flhDC</i> behind the different promoters, the purified PCR product was digested with <i>XbaI</i> and <i>PstI</i>, whereas the BioBrick plasmids were cut with <i>SpeI</i> and <i>PstI</i>. The digestion was performed as described in the <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a>. At first only three promoters were chosen: <br>
J23112 - 20E - x1 <br>
J23112 - 20E - x1 <br>
J23105 - 18M - x623 <br>
J23105 - 18M - x623 <br>
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{{GoettingenFooter}}

Latest revision as of 17:17, 22 September 2012

Deutsch  / English 

#2 Speed Improvement - 5th week

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V05_28


Miniprep of the distinct constitutive promoters
  • Experiment:
    Minipreps were done with PeqGOLD MiniPrep Kit (Peqlab) according to the user manual.



V05_29


V05_29_1 Preparative double digestion of flhDC and pUC18
  • Experiment:
    In order to clone flhDC behind the different promoters, the purified PCR product was digested with XbaI and PstI, whereas the BioBrick plasmids were cut with SpeI and PstI. The digestion was performed as described in the protocol. At first only three promoters were chosen:
    J23112 - 20E - x1
    J23105 - 18M - x623
    J23100 - 18 C - x 2547
    After the digestion the plasmids and the PCR product were loaded onto an 1% agarose gel and separated. Subsequently, the desired fragments were cut out and purified using peqGOLD Gel Extraction Kit (Peqlab) according to the manual.
  • Observations & Results:
    For all plasmids fragments of a size of 2.1 kb and 0.9 kb were obtained. Since these are the expected fragment sizes after a successful digestion the products can be used for further cloning.

V05_29_2 Insertion of flhDC into the BioBrick plamids
  • Experiment:
    The ligation of the digested flhDC with the BioBrick plasmids was conducted as described in the protocol.


V05_30


Chemical transformation of competent E. coli (DH10B)
  • Experiment:
    After the heat inactivation of the ligase at 65°C the ligated plasmids were chemically transformed into E. coli (DH10B) according to the standard protocol.
  • Observations & Results:
    The transformation was successful. No colonies have developd on the negative control plate whereas the positive control featured numerous. Also the plates with the desired constructs featured growth, however, on the 18C plate three red colonies could be observed. This, seems to be due to failed restriction or ligation.


V05_31


Preparation of over night cultures
  • Experiment:
    Over night cultures were preaped of three colonies of each constructs in order to isolate the plasmids.



V06_01


V06_01_1 Miniprep of the flhDC-promoter constructs
  • Experiment:
    Minipreps were conducted with PeqGOLD MiniPrep Kit (Peqlab) according to the user manual.

V06_01_2 Test digestion of the flhDC-promoter constructs
  • Experiment:
    All constructs were digested with XbaI and SpeI conducted as described in the protocol.
  • Observations & Results:
    For each construct two clones hosting the correctly inserted gene in the plasmid could be obtained. The others still contain the rfp gene and were thus rejected.


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