Team:UPNAvarra Spain/Notebook/Week4
Week 4
22nd July 2019
Trying to solve our problems with the PCR amplification, we decided to use a different Taq polymerase, from a new set, in order to discard a problem with the previous enzyme. Fortunately, we obtained a good result in this case. When we checked the amplified fragments on an agarose gel we confirmed that the size was as expected, so we followed with the DNA purification from the gel.
23rd July 2019
We continued with the construction of the plasmids. Regarding the nitrate construction, we digested the previously amplified fragment (promoter-RBS) (EcoRI-SpeI) and also the amil-CP-blue and amilGFP chromoproteins (EcoRI-XbaI), in order to performed the ligation and the electroporation.
In case of heavy metals, we had the different basic parts from the distribution kit prepared, so we started with the digestion of the promoter region (EcoRI-SpeI) and the RBS (EcoRI-XbaI), to continue with the ligation and the subsequent electroporation.
In case of heavy metals, we had the different basic parts from the distribution kit prepared, so we started with the digestion of the promoter region (EcoRI-SpeI) and the RBS (EcoRI-XbaI), to continue with the ligation and the subsequent electroporation.
24th July 2019
We spent this day checking the ligations. By colony PCR we could confirm that some constructions showed the appropriate size when checked on agarose gel. In that cases, we inoculate 3mL of LB medium with those colonies and left the culture growing overnight at 37°C.
25th July 2019
We performed the minipreps for the resulting ligations that showed the correct size (nitrate-amilCP, nitrate-amilGFP and mercury-RBS) and cross-checked them by digestion. When this was confirmed, we follow with another round of BioBrick assembly to add the next part to the constructions, the terminator in case of nitrate and the chromoproteins in case of heavy metals. So, we performed the corresponding digestions, ligations and electroporations.
With the remaining constructions (lead-RBS and arsenic-RBS), we checked more colonies by PCR traying to find successful ligations, but we did not be lucky enough to find anyone.
With the remaining constructions (lead-RBS and arsenic-RBS), we checked more colonies by PCR traying to find successful ligations, but we did not be lucky enough to find anyone.