Team:UPNAvarra Spain/Notebook/Week5
Week 5
29th July 2019
We obtained a lot of colonies from the previous ligations, and we checked them by colony PCR. Given the right result for the three of them (nitrate-amilCP-terminator; nitrate-amilGFP-terminator; mercury-RBS-tsPurple), we inoculated the positive colonies on LB medium.
For the remaining constructions, lead and arsenic, we repeated the ligation with the RBS sequence, changing the insert-vector ratio. After that, we electroporated the constructions.
For the remaining constructions, lead and arsenic, we repeated the ligation with the RBS sequence, changing the insert-vector ratio. After that, we electroporated the constructions.
30th July 2019
The digestion of the minipreps coming from the nitrate and mercury ligations showed us that the nitrate constructions, both with the amilCP and the amilGFP chromoproteins, had the expected size. So, we prepared the glycerol stock to keep the constructions for a long time.
We also had good results for mercury. In order to add the last part for this construction, the miniprep was digested, the ligation in a vector containing the terminator sequence was performed and the ligation was electroporated.
For lead and arsenic constructions, we performed a colony PCR of the colonies obtained after the promoter and RBS ligation. With positive results, we follow the cloning process picking positive colonies on LB liquid media and growing the cultures overnight.
We also had good results for mercury. In order to add the last part for this construction, the miniprep was digested, the ligation in a vector containing the terminator sequence was performed and the ligation was electroporated.
For lead and arsenic constructions, we performed a colony PCR of the colonies obtained after the promoter and RBS ligation. With positive results, we follow the cloning process picking positive colonies on LB liquid media and growing the cultures overnight.
31th July 2019
We followed with the electroporation of the mercury constructions and also, we performed some minipreps from the lead and arsenic ligation with the RBS sequence. When we checked the minipreps by digestion, the results were positive for both metals. Hence, we digested these constructions, performed the ligation with a chromoprotein (tsPurple for mercury and amilGFP for arsenic) and the electroporation.
1st August 2019
Hopefully, we obtained colonies from all our ligations, so only had to check some of them by colony PCR and confirm the sizes of the fragments to finish this week with a good taste in the mouth. Get on with it! Finally, we obtained positive results for mercury, but lead and arsenic constructions followed showing problems.