Team:Goettingen/week18-2

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#2 Speed Improvement - 18th week

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V08_27

V08_27_1 Miniprep of the tar-promoter constructs

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

V08_27_2 Chemical retransformation of the tar-promoter constructs into E. coli BL21

Experiment:
The retransformation was performed as described in the standard protocol.
Observations & Results:
The retransformation was successful since numerous colonies could be obtained except for the empty negative control.

V08_27_3 Chemical transformation of different motility increasing constructs into E. coli MG1655

Experiment:
For we aimed to investigate the effect of our constructs on cells that are used to swim competent MG1655 cells were transformed with the following constructs:
fliC (DH10B)
fliC (Salmonella)
motA
motB
yhjH

18M-flhDC
18O-flhDC
18C-flhDC
Observations & Results:
The transformation was not successful. The next morning only one plate (the positive control) featured any visible colonies. However, after several additional hours of incubation also some very small colonies appeared at some other plates. Nevertheless, the efficiency was very low. We suggest that this is normal since MG1655 is no cloning strain and might have thus a much lower transformation efficiency. This idea was supported by our inquiries.

V08_27_4 Preparation of over night cultures

Experiment:
In oder to perform a Real-Time Quantitative Reverse Transcription PCR over night cultures of the tar-promoter constructs in BL21 were prepared.

V08_28

Preparative double digestion of the flhDC-promoter constructs and pSB1C3

Experiment:
The flhDC-promoter constructs as well as the vector pSB1C3 still including tar were digested with EcoRI and PstI as described in the protocol. Afterwards, the fragments of the desired size were cut out and frozen in order to extract the DNA from the gel slices the following day.
Observations & Results:
The gel featured clearly visible bands of the expected length and thus the digestion was successful.

V08_29

Ligation and transformation of flhDC-promoter constructs and Psb1c3

Experiment:
TEXT

V08_30

Mini prep

Experiment:
TEXT

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 The transformation was not successful. The next morning only one plate (the positive control) featured any visible colonies. However, after several additional hours of incubation also some very small colonies appeared at some other plates. Nevertheless, the efficiency was very low. We suggest that this is normal since MG1655 is no cloning strain and might have thus a much lower transformation efficiency. This idea was supported by our inquiries. </li>
 <br> </ul>
-<b>V08_27_1 Preparation of over night cultures</b><br>
+<b>V08_27_4 Preparation of over night cultures</b><br>
 <ul>
 <li>Experiment: <br>
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 </table>
 <br>
 <table cellpadding="20 px" border="1" bordercolor="black" valign="top">
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 <td width="900" bordercolor="black" valign="top">
 <h2><b>V08_28 </b></h2><br>
-<b>Restriction of flhDC-promoter constructs and Psb1c3 </b><br>
+<b>Preparative double digestion of the <i>flhDC</i>-promoter constructs and pSB1C3</b><br>
 <ul>
 <li>Experiment: <br>
-TEXT</li>
+The flhDC-promoter constructs as well as the vector pSB1C3 still including <i>tar</i> were digested with EcoRI and PstI as described in the protocol. Afterwards, the fragments of the desired size were cut out and frozen in order to extract the DNA from the gel slices the following day. </li>
+<li>Observations & Results: <br>
+The gel featured clearly visible bands of the expected length and thus the digestion was successful. </li>
 </ul>
 <br></td></tr>