Team:Goettingen/week21-2

From 2012.igem.org

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Menu Bar 1
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Drop-Down Menu #2
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Side Menu #1
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div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM1,
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Drop-Down Menu #2
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Side Menu #2
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div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM2,
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Menu Bar 1
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Drop-Down Menu #2
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Side Menu #3
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div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM3,
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/*...............................................................................................*/
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<p></p><div id="header"><img src="http://www.patrickreinke.de/igem/headpicture_2.jpg" alt="Team Goettingen">
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Revision as of 10:14, 22 September 2012

Deutsch  / English 

#2 Speed Improvement - 21st week

Back to overview

V09_17


V09_17_1 Preparative double digestion of flhDC, FliC, 18K-RFP and pSB1C3 followed by ligation
  • Experiment:
    In order to clone the flhDC, FliC and 18K-RFP constructs into pSB1C3, all components were digested with EcoRI and PstI according to the protocol. The restriction products were subsequently separated on a gel in order to verify the digestions success and purify the desired fragments. After measuring the concentration of the purified fragments, an overnight ligation was initiated.

V09_17_2 Preparation of over night cultures
  • Experiment: We prepared overnight cultures of different promoter-gene constructs in two different E. coli strains (MG1655 & BL21) in order to test wether the motility is influenced. The following constructs were used:

    - 20E_flhDC
    - 18C_flhDC
    - 20I_flhdc
    - 20E_motA
    - 18C_motA
    - 20E_motB
    - 18C_motB


V09_18


V09_18_1 Motility Assays
  • Experiment: For the motility assay the over night cultures, with different constructs, were spun down and dropped onto M9- or 1% trypton-agar. The exact protocol you can find HERE
  • Observation and Results:
    Text

V09_18_2 Preparative double digestion of MotA, MotB, yhjH, 18C-RFP, 20E-RFP and 20I-RFP followed by ligation
  • Experiment:
    TEXT

V09_18_3 Chemical transformation of different plasmid-gene constructs into E. coli (DH10B) or (MG1655)
  • Experiment:
    Chemical transformation was done according to the standard protocol. The following constructs were used:

    - pSB1C3-FliC into E. coli (DH10B)
    - pSB1C3-flhDC into E. coli (DH10B)
    - PSB1C3-RFP into E. coli (MG1655)


V09_19


V09_19_1 Motility Assays
  • Experiment: For the motility assay the over night cultures, with different constructs, were spun down and dropped onto M9- or 1% trypton-agar. The exact protocol you can find HERE
  • Observation and Results:
    Text

V09_19_2 Chemical transformation
  • Experiment:
    For the chemical retransformation the standard protocol was followed. The following constructs were transformed:
    - pSB1C3-18C-motA (DH10B)
    - pSB1C3-18C-motB (DH10B)
    - pSB1C3-18C-yhjH (DH10B)
    - pSB1C3-20E-motA (DH10B)
    - pSB1C3-20E-motA (DH10B)
    - pSB1C3-20E-motA (DH10B)
    - pSB1C3-20I-motA (DH10B)
    - pSB1C3-20I-motB (DH10B)
    - pSB1C3-20I-yhjH (DH10B)
    - pSB1C3-RFP (BL21)


V09_19_3 Preparation of over night cultures
  • Experiment:
    Over night cultures were prepared of three colonies of each constructs in order to isolate the plasmid and check the correct insert.
    - pSB1C3-flhDC
    - pSB1C3-fliC
    - pSB1C3-18K-RFP


V09_20


V09_20_1 Mini Prep and test digestion of pSB1C3-gene constructs
  • Experiment:
    Minipreps were conducted with PeqGOLD MiniPrep Kit (Peqlab) according to the user manual.All constructs were digested with XbaI and SpeI conducted as described in the protocol.

    - pSB1C3-flhDC
    - pSB1C3-fliC
    - pSB1C3-18K-RFP

  • Observation and Results:
    All three the pSB1C3-18K-RFP clones host the correctly inserted gene in the plasmid. The pSB1C3-flhDC / fliC clones do not host the correctly inserted gene.

V09_20_2 PCR of fliC
  • Experiment:
    In order to get more fliC, the already quikchanged fliC was amplified via PCR. The PCR product was analyzed via gel-electrophoresis and subsequently purified using peqGOLD Gel Extraction Kit (Peqlab) according to the manual.

V09_20_3 Repetition of preparative double digestion of flhDC, FliC and pSB1C3
  • Experiment:
    In order to clone the flhDC, FliC constructs into pSB1C3, all components were digested with EcoRI and PstI according to the protocol.

V09_20_4 Preparation of over night cultures
  • Experiment:
    Over night cultures were prepared of three colonies of each constructs in order to isolate the plasmid and check the correct insert.

    - pSB1C3-18C-motA (DH10B)
    - pSB1C3-18C-motB (DH10B)
    - pSB1C3-18C-yhjH (DH10B)
    - pSB1C3-20E-motA (DH10B)
    - pSB1C3-20E-motA (DH10B)
    - pSB1C3-20E-motA (DH10B)
    - pSB1C3-20I-motA (DH10B)
    - pSB1C3-20I-motB (DH10B)
    - pSB1C3-20I-yhjH (DH10B)
    - pSB1C3-RFP (BL21)

V09_21


V09_21_1 Ligation and chemical Transformation of flhDC, FliC and pSB1C3
  • Experiment:
    The restriction products were separated on a gel in order to verify the digestions success and purify the desired fragments. After ligation of the genes into pSB1C3, we transformed the ligated plasmids into E. coli DH10B according to the protocol.


V09_21_2 Mini Prep and test digestion of pSB1C3-gene constructs
  • Experiment:
    Minipreps were conducted with PeqGOLD MiniPrep Kit (Peqlab) according to the user manual.All constructs were digested with XbaI and SpeI conducted as described in the protocol.

    - pSB1C3-18C-motA (DH10B)
    - pSB1C3-18C-motB (DH10B)
    - pSB1C3-18C-yhjH (DH10B)
    - pSB1C3-20E-motA (DH10B)
    - pSB1C3-20E-motA (DH10B)
    - pSB1C3-20E-motA (DH10B)
    - pSB1C3-20I-motA (DH10B)
    - pSB1C3-20I-motB (DH10B)
    - pSB1C3-20I-yhjH (DH10B)
    - pSB1C3-RFP (BL21)

  • Observation and Results:
    All three the pSB1C3-18K-RFP clones host the correctly inserted gene in the plasmid. The pSB1C3-flhDC / fliC clones do not host the correctly inserted gene.

V09_21_3 TITEL
  • Experiment:
    TEXT


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