Team:Goettingen/week6-2

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Revision as of 13:19, 18 September 2012

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Chemical retransformartion of the flhDC-promoter constructs into E. coli (DH10B)

Experiment:
For the chemical retransformation the standard protocol for transformation was followed. The following constructs were transformed into E. coli:
20E - #2
20E - #3
18M - #2
18M - #3
18C - #1
18C - #2
Observations & Results:
The retransformation was successful since all plates showed numeours colonoes except the negative control.

V06_05_1 Preparative double digestion of flhDC and plasmids containing further promoter

V06_05_2 Insertion of flhDC into the BioBrick plamids

Experiment:
The ligation of the digested flhDC with the BioBrick plasmids was conducted as described in the protocol.

Chemical transformartion of the flhDC-promoter constructs into E. coli (DH10B)

Experiment:
For the chemical transformation the standard protocol was followed.
Observations & Results:
The transformation was successful since all plates showed numeours colonoes except the negative control. However, on the plastes with 18K and 18O few red colonies could be observed. These were again rejected.

Preparation of over night cultures

Experiment:
Over night cultures were prepapred of three colonies of each constructs in order to isolate the plasmids.

V06_08_1 Miniprep of the new flhDC-promoter constructs

Experiment:
Minipreps were conducted with PeqGOLD MiniPrep Kit (Peqlab) according to the user manual.

V06_08_2 Test digestion of the new flhDC-promoter constructs
Experiment:
In order to prove correct insertion of flhDC a test digestion was performed using SpeI and XbaI according to the protocol.
Observations & Results:
For each construct two to three clones hosting the correctly inserted gene in the plasmid could be obtained. Those colonies still containing the rfp gene and were thus rejected.

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@@ Line 557: / Line 557: @@
 <ul>
 <li>Experiment:  <br>
-In order to clone <i>flhDC</i> behind further promoters, the purified PCR product was digested with XbaI and PstI, whereas the BioBrick plasmids were cut with SpeI and PstI. The digestion was performed as described in the protocol. This time the follwing promoters were used: <br>
+In order to clone <i>flhDC</i> behind further promoters, the purified PCR product was digested with <i>XbaI</i> and <i>PstI</i>, whereas the BioBrick plasmids were cut with <i>SpeI</i> and <i>PstI</i>. The digestion was performed as described in the protocol. This time the follwing promoters were used: <br>
 J23113 - 20G - x21 <br>
 J23109  - 2G - x106 <br>
@@ Line 622: / Line 622: @@
 <b>V06_08_2  Test digestion of the new <i>flhDC</i>-promoter constructs</b><br>
 <li>Experiment:  <br>
-In order to prove correct insertion of <i>flhDC</i> a test digestion was performed using SpeI and XbaI according to the protocol.<br>
+In order to prove correct insertion of <i>flhDC</i> a test digestion was performed using <i>SpeI</i> and <i>XbaI</i> according to the protocol.<br>
 <li>Observations & Results: <br>
 For each construct two to three clones hosting the correctly inserted gene in the plasmid could be obtained. Those colonies still containing the rfp gene and were thus rejected.</li>