Difference between revisions of "Team:Newcastle/Notebook/Lab/Lightbulb/Week 10"

August 24th 2016

We carried out the restriction digest protocol on our DNA using 10 uL of plasmid. However, instead of using NEBuffer2 we used CutSmart, therefore we added 5 uL of CutSmart. We didn’t use BSA or DpnI so our exact protocol differs from that stated in the above link. We used 0.5 uL of EcoRI-HF and PstI and then added 19 uL of dH2O. Once the Restriction Digest was complete, we carried out a Ligation and then placed all of our samples in the freezer to use during the following days.

August 25th 2016

We carried out a PCR clean up on our samples. While we followed the protocol originally, there were changes that were made. Therefore our Sample 1 needed 120uL of Binding solution and Sample 2 needed 65 uL of Binding Solution. Instead of using Elution Buffer we used JustWater because if it was TE, the EDTA may not work in the PR (bind the Mg). After the PCR Cleanup, we carried out a Gel Electrophoresis of the samples. Well 1 had the DNA Ladder, Well 2 had the plasmid Sample 1, Well 3 had the plasmid Sample 2 and Well 4 had another DNA ladder.

August 26th 2016

Using a Quibit Fluorimeter, we measure the concentration of plasmid pSB1C3 post clean-up for ligation. We then freeze dried and spun down the samples which allowed us to quickly dry and concentrate our small sample volumes. We then used 11 uL of this and diluted to 16.5 uL for the Digestion protocol which we used on the 24/08/2016.