Team:Goettingen/week17-2

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#2 Speed Improvement - 17th week

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V08_20

Preparative double digestion of pSB1C3

Experiment:
The vector pSB1C3 in its linearized as well as in its circularized form was 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 bands of the expected sizes when irradiated with UV rays.

V08_21

Purification of the flhDC-promoter constructs

Experiment:
The restricted vector was purified using peqGOLD Gel Extraction Kit (Peqlab) according to the manual.
Observations & Results:
After the purification the DNA concentration was measured with the NanoDrop according to which our samples did not contain any DNA. This is also right for the purified flhDC-promoter constructs. Since NanoDrops are not very accurate, the samples were additionally loaded on a 1% agarose gel, which also did not show any bands expect for those of the marker. Since we already have had problems to receive proper DNA concentration with our purification kit and both preparative gels showed the expected band, we assume that the DNA got lost during purification.

V08_22

Chemical transformation of the eight flhDC-promoter constructs into E. coli BL21

Experiment:
In order to receive fresh colonies for the subsequent preparation of glycerol stocks competent E. coli BL21 cells were transformed with the eight flhDC-promoter constructs. The other strains were neglected for BL21 showed the strongest susceptibility to inserted constructs.
Observations & Results:
The transformation was successful since all plates showed numerous colonies except for the negative control.

V08_23

Preparation of overnight cultures

Experiment:
In order to prepare the glycerol stocks over night cultures of the eight flhDC-promoter constructs in E. coli BL21 were prepared. Additionally, over night cultures of MG1655 were prepared for a subsequent preparation of competent cells.

V08_13

V08_24_1 Preparation of glycerol stocks of the eight flhDC-promoter constructs in E. coliBL21 and the mutation library

Experiment:
The glycerol stocks were prepared according to the following protocol. Furthermore, glycerol stock of the mutation library constructed by group 3 were prepared.

V08_24_2 Chemical transformation of the tar-promoter constructs into E. coli BL21

Experiment:
For the chemical transformation the standard protocol was followed.
Observations & Results:
The transformation was successful since numerous colonies were obtained except for the negative control.

V08_14

Performance of motility assay

Experiment:
Here again methionine was added to the prepared M9 agar plates and the Whatman Filter Paper were imbued with an amino acid mixture solution as an attractant. The over night cultures were dropped onto the plates directly.
Observations & Results:
Two days after motility assay preparation (16th of August) first stirrings of swimming were visible. In general BL21 shows the strongest motility followed by DH10B. XL1 Blue and DH5α are rather poor. The strongest effect can be attributed to fliC (especially the gene originating from DH10B) and motB. However, the comparison to the reference pUC18 is for all strains rather disillusioning. Whereas pUC18 lead to rather small halos in comparison to fliC and motB at some plates, on other plates pUC18 caused the biggest ones. Thus, the results cannot unambiguously be evaluated.

V08_15

Preparation of over night cultures

Experiment:
In order to reproduce the motility assay from the day before new over night cultures of fliC (DH10B), fliC (Salmonella), motA, motB, yhjH and pUC18 in E. coli DH10B, BL21, DH5α and XL1 Blue were prepared.
Observations & Results:
The growing of the culture again posed a problem to us since some cultures grew quite nice, whereas others multiplied only poorly to not at all. Therefore, we were not able to conduct the assay.

V08_16

Preparative double digestion of 18M-flhDC, 18O-flhDC and 18C-flhDC

Experiment:
The flhDC-promoter constructs were digested with XbaI 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 sizes when irradiated with UV rays.

V08_17

Purification of the flhDC-promoter constructs

Experiment:
The restriction products were purified using peqGOLD Gel Extraction Kit (Peqlab) according to the manual.

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@@ Line 589: / Line 589: @@
 <h2><b>V08_13 </b></h2><br>
-<b>V08_13_1  Performance of motility assay </b><br>
+<b>V08_24_1  Preparation of glycerol stocks of the eight <i>flhDC</i>-promoter constructs in <i>E. coli</i>BL21 and the mutation library </b><br>
 <ul>
 <li>Experiment: <br>
-This time methionine was added to the prepared M9 agar plates and the Whatman Filter Paper were imbued with an amino acid mixture solution as an attractant. Here again the over night cultures were dropped onto the plates directly.  <br></li>
+The glycerol stocks were prepared according to the following protocol. Furthermore, glycerol stock of the mutation library constructed by group 3 were prepared. <br></li>
-<li>Observations & Results: <br>
-The following day (14th August) already swimming could be observed on all plates. In general MG1655 featured the strongest motility, followed by <i>tar</i>-18C. This pattern intensified over the following days, but chemotactic behavior could not clearly be identified at all.</i></ul>
 <br>
-<b>V08_13_2 Preparation of over night cultures </b><br>
+<b>V08_24_2 Chemical transformation of the <i>tar</i>-promoter constructs into <i>E. coli</i> BL21  </b><br>
 <ul>
 <li>Experiment:  <br>
-For we aimed to test <i>fliC</i> (DH10B), <i>fliC</i> (<i>Salmonella</i>), <i>motA</i>, <i>motB</i> and <i>yhjH</i> again, over night cultures of the constructs in <i>E. coli</i> DH10B, BL21, DH5&alpha; and XL1 Blue were prepared. Furthermore, cultures of pUC18 in the different strains as a reference were made.<br>
+For the chemical transformation the standard protocol was followed.<br></li>
+<li>Observations & Results: <br>
+The transformation was successful since numerous colonies were obtained except for the negative control.
+</li>
 </ul><br>