Team:UPNAvarra Spain/Notebook/Week1
Week 1
1st July 2019
To culture our bacteria, we prepared LB growth media, by dissolving LB in deionized water. Then we added agar to create a gel for bacteria to grow upon after cooling down. After autoclaving, we allowed the LB media to cool to 55°C and afterwards, we added the antibiotic (chloramphenicol 20 mg/L) and poured the plates under a sterile environment. We also prepared liquid media.
2nd July 2019
We checked the efficiency of the competent cells using the kit provided by iGEM for that purpose. We followed the instructions of the kit, and electroporated a plasmid harboring the RFP construct at two different concentrations.
At the same time, we electroporated a plasmid from the Distribution Kit, composed of a nitrate sensitive promoter and the GFP protein as a reporter.
At the same time, we electroporated a plasmid from the Distribution Kit, composed of a nitrate sensitive promoter and the GFP protein as a reporter.
3rd July 2019
We obtained the results of our competent cells efficiency, counting colonies and calculating the cfu/µg DNA. So, we could confirm that they were perfectly working. We also obtained some colonies in the plate from the transformation of the plasmid coming from the kit. We inoculate tubes containing 3 mL of LB medium with a single colony for each one and we left them growing overnight at 37ºC shaking.
4th July 2019
We performed the minipreps and digested the plasmid with restriction enzymes (EcoRI-PstI). After a gel electrophoresis we visualized the size of the fragments under UV light and we could confirm that they corresponded with the expected sizes. We prepared the glycerol stock for this construction.