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#2 Speed Improvement - 9th week

V06_25_1 Chemical transformation of the BioBrick plasmid pSB1C3 into E. coli (DH10B)

Experiment:
In order to gain further plasmid material of the new vector pSB1C3, it was transformed into E. coli (DH10B) as described in the protocol.
Observations & Results:
The transformation failed. Only the positive control featured colony formation. We found out that accidentally ampicillin containing plates were used whereas the plasmid obtains a chloramphenicol resistance. Thus, the result is not surprising.

V06_25_2 Preparation of over night cultures

Experiment:
Since we received a new E. coli (DH10B) strain, over night cultures were prepared in order to produce new competent cells. The following protocol was considered.

V06_26_1 Preparation of chemically competent cells of the new E.coli (DH10B) strain

Experiment:
The preparation of the competent cells was performed according to the following protocol.
Observations & Results:
The performance of a test transformation proved the functionality of the new competent cells.

V06_26_2 Preparation of over night cultures

Experiment:
In order to test different E. coli strains competent cells of the recently received strains BL21, DH5α and XL1 Blue should be produced. On that account over night cultures of al strains were prepared.

V06_27_1 Preparation of chemically competent cells of the new E.coli strains BL21, DH5α and XL1 Blue

Experiment:
The preparation of the competent cells was performed according to the following protocol.

V06_27_2 Chemical transformation of flhDC-promoter constructs into E.coli (DH10B, BL21, DH5α and XL1 Blue)

Observations & Results:
The transformation of the DH10B cells functioned very well. Numerous colonies could be found at all plates except for the negative control. The other three strains did not lead to such good results. Here, no or low bacterial growth could be observed. Since the competent cells of DH10B worked so well, we suggested that the other strains were just too fresh, so the transformation should be repeated.

V06_28_1 Repetition of the chemical transformation of flhDC-promoter constructs into E.coli (BL21, DH5α and XL1 Blue

Observations & Results:
The transformation of BL21, DH5α and XL1 Blue was successful this time. All plates featured numerous colonies except the negative controls.
Observations & Results:
The transformation of the DH10B cells functioned very well. Numerous colonies could be found at all plates except for the negative control. The other three strains did not lead to such good results. Here, now or low bacterial growth could be observed. Since the competent cells of DH10B worked so well, we suggested that the other strains were just too fresh, so the transformation should be repeated.

V06_28_2 Chemical transformation of pSB1C3 into E.coli (DH10B)

Experiment:
For the chemical transformation the standard protocol was considered. The plasmid was used in its uncut form as well as restricted.
Observations & Results:
The transformation with the uncut DNA was successful whereas that using the restricted plasmid failed. This seems to be due to the fact that the restricted vector had not been religated by the cells.

V06_28_3 Preparation of over night cultures

Experiment:
Over night cultures of the four strains DH10B, BL21, DH5α and XL1 Blue hosting the three flhDC-promoter constructs were prepared.
Observations & Results:
The inoculation of the over night cultures failed. Perhaps something was wrong with the shaker.