Team:Newcastle/Notebook/Lab/Lightbulb/Week 11

August 30th 2016

Ligation was carried out with the purified PCR plasmid from the previous week. We had 2 replicates of our 'lightbulb' and 'battery' constructs. Our DNA2.0 sample was in the form of filter paper. Therefore we used half of the filter paper in case something went wrong. We placed it on a sterile surface (petri dish lid inside the hood) and added 50 uL of water directly to the filter paper. We then incubated it at room temperature for 2 minutes. We punctured a 0.5 mL tube using a syringe and placed the filter paper into the tube. We then placed the 0.5 mL tube into a larger 1.5 mL tube. We centrifuged it at 14,000 rpm for 1 minute and then discarded the 0.5 mL tube with the filter paper. According to DNA2.0 we have approximately 45 uL of buffer and DNA. In the afternoon we carried out the transformation of our cells. The protocol can be found here. We plated our samples and let them grow overnight.

August 31st 2016

Made agar for LB plates and autoclaved overnight. The agar recipe we used was 7 g of Agar, 10 g of LB and 400 mL of H₂O. In the meantime we grew up liquid cultures of DNA2.0. Some of the plates we grew up had very small colonies on them and therefore we left them to grow some more so that the colonies could become a bit larger.

September 1st 2016

We made up some more agar plates and re-plated our sample due to the background colonies present in previous plates. We made 22 plates. We then grew up liquid cultures from the plates. Matt said that in liquid, the antibiotic works better (bigger surface area) and therefore it should select for our constructs better. We will mini-prep these tomorrow.