Team:Goettingen/week19-2

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<h2><b>V09_03 </b></h2><br>
<h2><b>V09_03 </b></h2><br>
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<b>Quikchange/overlapping PCR of <i>fliC</i></b><br>
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<b>Overlapping PCR of <i>fliC</i></b><br>
<ul>
<ul>
<li>Experiment: <br> In order to eliminate undesired restriction sites inside of our <i>fliC</i> part a Overlapping PCR was performed using especially designed primers that will cause a point mutation and <i>PfuTurbo</i> polymerase (<a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a>). Since four unwanted restriction sites are localized in our part four reactions were prepared producing overlapping fragments. Subsequently samples 1 and 2 as well as 3 and 4 were combined via overlapping PCR reducing the number of fragments at two. Finally sample 1+2 and 3+4 were combined as well and one final DNA fragment is obtained. <br></li>
<li>Experiment: <br> In order to eliminate undesired restriction sites inside of our <i>fliC</i> part a Overlapping PCR was performed using especially designed primers that will cause a point mutation and <i>PfuTurbo</i> polymerase (<a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a>). Since four unwanted restriction sites are localized in our part four reactions were prepared producing overlapping fragments. Subsequently samples 1 and 2 as well as 3 and 4 were combined via overlapping PCR reducing the number of fragments at two. Finally sample 1+2 and 3+4 were combined as well and one final DNA fragment is obtained. <br></li>

Revision as of 11:33, 22 September 2012

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#2 Speed Improvement - 19th week

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V09_03


Overlapping PCR of fliC
  • Experiment:
    In order to eliminate undesired restriction sites inside of our fliC part a Overlapping PCR was performed using especially designed primers that will cause a point mutation and PfuTurbo polymerase (protocol). Since four unwanted restriction sites are localized in our part four reactions were prepared producing overlapping fragments. Subsequently samples 1 and 2 as well as 3 and 4 were combined via overlapping PCR reducing the number of fragments at two. Finally sample 1+2 and 3+4 were combined as well and one final DNA fragment is obtained.
  • Observations & Results:
    The QC PCR was not successful. We were not able to observe any PCR product.


V09_04


V09_04_1 Test digestion of pSB1C3 (including flHDC under the control of different promoters)
  • Experiment:
    The test digestion of pSB1C3 including flHDC under the expression of 8 different promoters was performed with the restriction enzymes EcoRI and PstI according to the protocol. The product was subsequently analyzed on an 1% analytical agarose gel.
  • Observations & Results:
    Since fragments of the expected size could be obtained for all clones the test digestion worked for all.

V09_04_2 Biobrick Standardization of motA,motB and yhjH
  • Experiment:
    PCR of motA, motB and yhjH using primers containing the standard BioBrick sequences. Procedure was succesful and the PCR products were purified via PeqGOLD Gelextraction Kit (Peqlab).


V09_05


V09_05_1 Preparative double digestion of motA, motB, yhjH and pSB1C3
  • Experiment:
    motA, motB, yhjH and pSB1C3 were digested with EcoRI and PstI. The prodcuts were seperated via gel electrophoresis and purified using PeqGOLD Gelextraction Kit (Peqlab).

V09_05_2 Repetition of the Overlapping PCR of fliC
  • Experiment:
    Here again the Overlapping PCR was performed using designed primers and PfuTurbo polymerase (protocol). However, this time a lower annealing temperature was chosen. Also this time four short fragments were produced first and subsequently combined to a complete final DNA fragment.
  • Observations & Results:
    Once again the QC PCR failed for no PCR product was observed. Since we already had problems with enzymes during our experiments we consider an non-functionable polymerase.


V09_06


V09_06_1 Ligation of motA, motB and yhjH into pSB1C3
  • Experiment:
    The ligation of motA, motB and yhjH with pSB1C3 was conducted as described in the protocol.

V09_06_2 Chemical transformation of motA, motB and yhjH to E. coli DH10B
  • Experiment:
    The transformation was performed as described in the standard protocol.
  • Observations & Results:
    The transformation was successful since numerous colonies has formed whereas the negative control remained clear.


V09_09


Preparation of over night cultures
  • Experiment:
    Over night cultures were prepared:

    pSB1C3-motA
    pSB1C3-motB
    pSB1C3-yhjH



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