Team:Goettingen/week5-3

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(Difference between revisions)
 
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<b>Insertion of <i>tar</i> into pUC18</b><br>
<b>Insertion of <i>tar</i> into pUC18</b><br>
<ul>
<ul>
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<li>Experiment: <br>Double digest of <i>tar</i> and pUC18 using <i>Eco</i>RI, <i>Pst</i>I and Orange buffer (ThermoScientific) according to <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a>. Gel extraction and purification of pUC18 and <i>tar</i> using peqGOLD Gelextraction Kit (Peqlab). Ligation of <i>tar</i> into pUC18 using T4 DNA Ligase (Thermo Scientific).  
+
<li>Experiment: <br>Double digest of <i>tar</i> and pUC18 using <i>Eco</i>RI, <i>Pst</i>I and Orange buffer (ThermoScientific) according to <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a>. Gel extraction and purification of pUC18 and <i>tar</i> using peqGOLD Gel Extraction Kit (Peqlab). Ligation of <i>tar</i> into pUC18 using T4 DNA Ligase (Thermo Scientific).  
</li>
</li>
</ul>
</ul>
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<b>Changing the <i>Xba</i>I site in the <i>tar</i> sequence via QuikChange</b><br>
<b>Changing the <i>Xba</i>I site in the <i>tar</i> sequence via QuikChange</b><br>
<ul>
<ul>
-
<li>Experiment: <br>DNA was prepped using peqGOLD MiniPrep Kit (Peqlab). A DNA test digest with <i>Eco</i>RI and <i>Xba</i>I was performed according to <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a> and analyzed on a 1% agarose gel. Test digest showed 8 out of 10 clones contained the construct pUC18_TAR. QuikChange PCR was performed using designed primers and <i>Pfu</i> Turbo polymerase (see QuikChange protocol <a href="https://2012.igem.org/Team:Goettingen/Project/Methods#QuikChange_Protocol">here</a>). Resulting construct was labeled pUC18_TAR_QC.
+
<li>Experiment: <br>DNA was prepped using peqGOLD Miniprep Kit (Peqlab). A DNA test digest with <i>Eco</i>RI and <i>Xba</i>I was performed according to <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a> and analyzed on a 1% agarose gel. Test digest showed 8 out of 10 clones contained the construct pUC18_TAR. QuikChange PCR was performed using designed primers and <i>Pfu</i> Turbo polymerase (see QuikChange protocol <a href="https://2012.igem.org/Team:Goettingen/Project/Methods#QuikChange_Protocol">here</a>). Resulting construct was labeled pUC18_TAR_QC.
</li>
</li>
</ul>
</ul>

Latest revision as of 11:24, 22 September 2012

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#3 Chemoreceptor Library - 5th Week

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V05_29


Insertion of tar into pUC18
  • Experiment:
    Double digest of tar and pUC18 using EcoRI, PstI and Orange buffer (ThermoScientific) according to protocol. Gel extraction and purification of pUC18 and tar using peqGOLD Gel Extraction Kit (Peqlab). Ligation of tar into pUC18 using T4 DNA Ligase (Thermo Scientific).


V05_30


Transformation of DH10B with pUC18_TAR construct
  • Experiment:
    Transformation was carried out according to protocol. Transformed DH10B were plated on LB-Amp plates (see here for recipe). Transformation was successful.


V05_31


Preparation of pUC18_TAR
  • Experiment:
    Inocculation of overnight cultures of 10 supposedly positive clones in liquid LB-Amp (recipe).


V06_01


Changing the XbaI site in the tar sequence via QuikChange
  • Experiment:
    DNA was prepped using peqGOLD Miniprep Kit (Peqlab). A DNA test digest with EcoRI and XbaI was performed according to protocol and analyzed on a 1% agarose gel. Test digest showed 8 out of 10 clones contained the construct pUC18_TAR. QuikChange PCR was performed using designed primers and Pfu Turbo polymerase (see QuikChange protocol here). Resulting construct was labeled pUC18_TAR_QC.


V06_02


Changing the XbaI site in the tar sequence via QuikChange
  • Experiment:
    DpnI digest of QuikChange PCR product to get rid of the parent plasmid.


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