Team:Goettingen/week20-2

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Revision as of 11:42, 18 September 2012

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V09_10_1 Miniprep of pSB1C3 (including motA, motB or yhjH)

Experiment:
Plasmids were prepped using PeqGOLD MiniPrep Kit (Peqlab) accirding to the manual.

V09_10_2 Test digestion of pSB1C3 (including motA, motB or yhjH)

Experiment:
Test digestion of pSB1C3 (including motA, motB or yhjH) with the restriction enzymes EcoRI and PstI. Afterwards, the product was loaded in a 1% analytical gel in order to verify its success.
Observations & Results:
The digestion worked for all clones since fragments with the appropriate size were produced over all.

V09_10_3 Digestion of motA, motB, yhjHand the promoters 20E, 20I and 18C

Experiment:
motA, motB or yhjH were digested with XbaI and PstI. The plasmids with the different promoters were treated with SpeI and PstI. The digestion was then controlled via gel-electrophoresis.
Observations & Results:
Since for all clones band of the expected size could be obtained these were isolated from the gel and purified using PeqGOLD Gelextraction Kit (Peqlab) according to the manual.

V09_10_4 Quickchange of fliC

Experiment:
Quick Change overlapping PCR was performed using designed primers and Pfu Turbo polymerase (see Quick Change protocol in "protocols").
Observations & Results:
Once again the QC PCR failed for no PCR product was observed.

V09_11_1 Insertion motA, motB and yhjH into J61002

V09_11_3 Quickchange of fliC part 1

V09_12_1 Quickchange of FliC part 2

V09_12_2 Restriction and Ligation of promoter-RFP constructs into pSB1C3

V09_13_1 Transformation of Psb1c3-Promoter-RFP constructs into e. coli DH10B

V09_13_2 Restriction and Ligation of FliC

V09_13_3 Preparation of over night cultures

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 <td width="900" bordercolor="black" valign="top">
 <h2><b>V09_11 </b></h2><br>
-<b>V09_11_1 Ligation and Transformation of <i>motA</i>, <i>motB</i> and <i>yhjH</i></b><br>
+<b>V09_11_1 Insertion <i>motA</i>, <i>motB</i> and <i>yhjH</i> into J61002 </b><br>
 <ul>
-<li>Experiment: <br>  After the ligation of <i>motA</i>, <i>motB</i> and <i>yhjH</i> into the plasmid J61002 with different promoters (20E, 20I and 18C), the ligation products were transformed into the <i>E. coli</i> strain DH10B.</li>
+<li>Experiment: <br>  <i>motA</i>, <i>motB</i> and <i>yhjH</i> were ligated nto the plasmid J61002 with different promoters (20E, 20I and 18C) according to the protocol.
+<b>V09_11_2 Chemical transformation of <i>motA</i>, <i>motB</i> and <i>yhjH</i> into  <i>E. coli</i> DH10B </b><br>
+<ul>
+<li>Experiment: <br>
+The ligation products were transformed into the <i>E. coli</i> DH10B as described in the standard protocol.</li>
+<li>Observations & Results: <br>
+The transformation was successful since numerous colonies were grown on all plates except for the negative control.</li>
+</ul>
 </ul>
 <br>
-<b>V09_11_2 Quickchange of <i>fliC</i> part 1</b><br>
+<b>V09_11_3 Quickchange of <i>fliC</i> part 1</b><br>
 <ul>
 <li>Experiment: <br> TEXT