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Team:Wageningen UR/Notebook/Proj1/June
From 2011.igem.org
June 1
12 colonies were screened by colony PCR: I0462 R0090 K325909 K325210 R0062 R0063
A BBa_10462 transformant was used as a positive control and plasmids of BBa_10462 were used in the PCR.
The PCR was confirmed for some of the samples. The corresponding colonies (BBa_10462, -K325210, -E0422, -C0062 and -P0412 transformants) inoculated 10 mL LB medium with Amp and were incubated on a shaker at 37°C overnight.
June 2
With colony PCRs, transformed colonies were found with: BBa_1712019, -"K325210" and -K325219. No transformed colonies were found that should contain: BBa_R0040, -K325909, -R0062 or -R0063. The former colonies were grown in 10 mL LB medium in presence of Amp on a shaker at 37°C overnight.
All inoculations of June 1, but one: the BBa_K325210 transformants, yielded enough growth. The specific colony was incubated, in the same way, again. 5 mL of each of the other cultures was used in miniprepping.
June 3
Of all incubations, only cultures of BBa_1712019 transformants have arisen. These transformants were plated on LB medium with Amp.
The yield of miniprepping was determined by using the Nanodrop spectrophotometer. The plasmids were present in the following concentrations:
- 95.4 ng/µL of BBa_C0062;
- 61.0 ng/µL and 70.5 ng/µL of BBa_E0422;
- 86.2 ng/µL of BBa_I0462 and
- 86.2 ng/µL of BBa_P0412.
June 6
On June 3 was seen BBa_"K325210" and -K325219 transformants did not grow. These have been incubated in the same way as on June 2 again. Also the colony PCR determined transformants with BBa_10462, -E0422, -C0062 and -P0412 in liquid culture (see June 1) were incubated again (BBa_K325210 transformants have not shown growth until this moment). Finally, on June 2 no transformants with BBa_R0062 or -R0063 were found; these have been incubated again too.
June 7
The liquid cultures were inspected: All incubations resulted in growth. 5 mL of each of these cultures have been used for miniprepping with the Fermentas GeneJET Miniprep kit. Two transformants still had to be found, which contained BBa_R0040 and -K325909 (see June 2). New colonies were picked out for colony PCRs and inoculated LB medium in an Eppendorf tube. Afterwards, an electrophoresis gel showed a BBa_R0040 transformant was found. Subsequently 1 µL of the inoculated medium was added to 10 mL of LB medium that included Amp.
Next to this, transformations with the introduced BioBrick parts BBa_C0261, -C0060, -S0168, -B0034, -B0015, -C0061 and -F2621 were performed. In this, a heat shock of 42°C was applied for 1 minute, 250 µL SOC medium was added and the Top10 competent cells were incubated on a shaker at 37°C during 1 hour. 50 µL of the medium was added to Amp containing plates A and 250 µL to Amp containing plates B. The medium of supposed BBa_B0015 transformants inoculated plates that contained both antibiotics Amp and Kan.
June 8
The plasmids that came out of the miniprepping of the clones incubated on June 6 were digested with restriction enzymes. Both a single digestion with ecoRI and a double digestion with ecoRI and speI together were carried out. The result of this was evaluated by a PCR: The digestions had succeeded. Fresh cultures, in LB medium holding Amp, were made of the clones with on of the following BioBrick parts: BBa_R0040, -K325210, -"K324210", -I712019, -I0462, -E0422, -K325219, -R0062 and -R0063. From the liquid culture made on June 7 the BBa_R0040 plasmids were isolated. Furthermore colony PCR tested a colony from the culture and the rest of the colony was transferred to an Eppendorf tube and it later inoculated a plate.
Top10 cells with BBa_C0261, -C0061, -C0060 and –F2621, out of the transformation on June 7, were screened with a colony PCR in which the time of the elongation step in each cycle was reduced to 1 min. Competent cells grown on Amp, Cm and Kan were used in the negative control of it. The colony PCR confirmed transformations, these clones inoculated 10 mL LB containing Amp and were incubated overnight.
June 9
The plasmids in all liquid cultures that were made on June 8, except for the supposed BBa_K325219 and -K32510 transformed Top10 cells, were evaluated with PCRs. Of the two exceptions other cultures were made in LB medium that contained the antibiotic Cm. Above this, the overnight incubated BBa_C0261, -C0061, -C0060 and –F2621 clones were miniprepped and analyzed by PCRs. Also expected BBa_B0034 and -B0015 clone colonies were screened by colony PCR. Not included the BBa_I742019, all BioBrick parts were confirmed by the PCRs. Liquid cultures were made of the corresponding clones. In the end the concentrations of isolated plasmids were determined by using the Nanodrop spectrophotometer.
June 10
Restriction analyses were performed on the liquid cultures mentioned on June 9. This was not the case for the BBa_B0034 and -B0015 clones though; these were miniprepped and the succes of these determined by the Nanodrop spectrophotometer. In the way as performed on June 7, new BioBrick parts were introduced. Plated on ampicillin rich medium are the with BBa_S03119 and -K08202 transformed Top10 cells.
