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V09_03
Quikchange of fliC
- Experiment:
In order to eliminate undesired restriction sites inside of our part a Quikchange PCR was performed using especially designed primers that will cause a point mutation and PfuTurbo polymerase (see Quick Change protocol in "protocols"). 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 smaple 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.
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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).
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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 Quikchange of fliC
- Experiment:
Here again the Quikchange PCR was performed using designed primers and PfuTurbo polymerase (see Quick Change protocol in "protocols"). 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.
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V09_06
V09_06_1 Insertion 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.
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V09_09
Preparation of over night cultures
- Experiment:
Over night cultures were prepared:
pSB1C3-motA
pSB1C3-motB
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