Team:UPNAvarra Spain/Notebook/Week3
Week 3
15th July 2019
We started preparing culture media, liquid and solid (plates) with the corresponding antibiotic, chloramphenicol. Then, we transformed different parts from the Distribution kit by electroporation, and also grew the nitrate-GFP construction on a plate from our glycerol stock.
16h July 2019
In this session we obtained multiple colonies from the electroporation of the parts from the kit, so we decided to analyze some colonies from each one, inoculating LB medium with the colony and an appropriate antibiotic, and growing the culture overnight at 37°C.
We followed working in parallel with the nitrate-GFP construction, just to verify that the promoter was working. So, we launched a preinoculum.
At the same time, we performed a PCR using the nitrate-GFP construction as template in order to amplify the promoter and the RBS region, adding the prefix and suffix BioBricks sequences.
For the PCR, we prepared our primers to reach a concentration of 10 pmol/µLr. We prepared the PCR master mix with water, primers, nucleotides, DNA template (1:100 miniprep) and the Taq polymerase. After that, we checked the PCR products by agarose gel electrophoresis, however we realized that there were no amplification.
We followed working in parallel with the nitrate-GFP construction, just to verify that the promoter was working. So, we launched a preinoculum.
At the same time, we performed a PCR using the nitrate-GFP construction as template in order to amplify the promoter and the RBS region, adding the prefix and suffix BioBricks sequences.
For the PCR, we prepared our primers to reach a concentration of 10 pmol/µLr. We prepared the PCR master mix with water, primers, nucleotides, DNA template (1:100 miniprep) and the Taq polymerase. After that, we checked the PCR products by agarose gel electrophoresis, however we realized that there were no amplification.
17th July 2019
We performed minipreps from the different cultures. After that, given the bad results obtained from the PCR amplification, we repeated the process with changes in the annealing temperature. Nevertheless, we did not obtain good results.
At the same time, we continued with the nitrate-GFP construction. We performed an induction with different KNO3– concentrations and, we checked the fluorescence emission of the bacterial resulting pellets under UV light.
At the same time, we continued with the nitrate-GFP construction. We performed an induction with different KNO3– concentrations and, we checked the fluorescence emission of the bacterial resulting pellets under UV light.
18th July 2019
Firstly, we checked the minipreps of the different parts by digestion with restriction enzymes. We could corroborate that the resulting fragments were in accordance with the expected sizes, so we prepared the glycerol stock for the most of them.
About our PCR attempts, we decided to repeat the amplification with a different pair of primers, but finally the result remained being negative.
About our PCR attempts, we decided to repeat the amplification with a different pair of primers, but finally the result remained being negative.