Team:Wageningen UR/Notebook/Proj1/May

May 3

Meeting with modeling person Hans Stigter from Biometris.

May 17

Competent E. coli TOP10 cells were transformed with deletion plasmids, by a heat shock of 42°C during 1 min.

May 20

50 µL of competent E. coli TOP10 cells were transformed with 1.5 µL solutions of the BioBrick parts in duplo, by incubation on ice for 15-40 min. followed by a heat shock at 42°C during 1 min. The following parts had to be inserted: BBa_I0460, -F2621, -F1610, -K325219, -K325210, -PO412 and -K325909. 250 µL SOC medium was added and they were incubated for 1.5 hour at 37°C (300 rpm). 50 µL of these inoculated plates A, 250 µL of these inoculated plates B.

May 23

Of the Top10 BBa_F2621 and BBa_K325219 transformants, which were made on May 20, a few colonies were found. More colonies were found of the BBa_K325210 and BBa_PO412 transformants. The rest of the transformations has not yielded growth.

May 24

Following the protocol of May 20, E. coli were transformed with BBa_F2621 (marker: Amp), -325219 (marker: Chl), -325210 (marker: Chl), -F1610 (marker: Kan), -K325909 (marker: Chl) and -I0460 (marker: Kan).

By colony PCR some of colonies were screened. The Invitrogen protocol was used. The PCR thermocycler, of Biorad, was set to the following values for 30 cycles: 94°C at the denaturation, 50°C at the annealing and 72°C at the elongation step. Of the following transformants 4 colonies were taken: BBa_F2621, -32519, -32510, -E0422 and -I0462.

The 4 colonies BBa_E0422 transformants and 3 colonies of the BBa_I0462 transformants were confirmed. For the rest of the samples the electrophoresis gel was not clear.

Liquid cultures of the BBa_E0422 and BBa_I0462 transformants were made. Tubes with 10 mL of LB medium and 10 µL Amp (stock) were inoculated with the corresponding colonies and untransformed Top10 cells - as a negative control. These were incubated over night.

May 25

The liquid cultures were made successfully on May 24 - unlike the other incubations, the negative control showed no growth.

With colony PCR the cultures were screened. The Biorad PCR thermocycler was set up in the following way: 30 cycles of 94°C at the denaturation, 50°C at the annealing and 72°C at the elongation step (duration of it was 2.5 min. because the length the luciferase gene is around 2600 bp).

May 30

PCRs were perfomed on BBa_I0462 transformants in liquid culture and colonies, a BBa_I0462 plasmid and a control.

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