Difference between revisions of "Team:Newcastle/Notebook/Lab/Lightbulb/Week 13"

September 12th 2016

Before ligation we had to work out how much DNA we had after our restriction digest. We used the Quibit Fluorimeter again with n=6 and 2uL. We used the dsDNA broad range. We finalized that for the Battery we need 16.09ng and for the VR we need 5.652 ng. We left the samples on the bench for 30 minutes to give the reagent more time to react because the values obtained were still quite small. After the 30 minutes the results were:

• Battery 1 – 3.52 uL/mL
• Battery 2 – 4.02 uL/mL
• VR 1 – 38.2 ug/mL
• VR 2 – 26.0 ug/mL

Using these samples as well as NEBiocalculator.neb.com, we were able to calculate how much DNA we had. For Battery 1 and 2 we had 5.63uL and 4.33 uL accordingly. For VR 1 and 2 on the other hand through we had 0.171 and 0.239 uL accordingly. These values were very small so we had to carry out a 1 in 5 dilution. We then carried out the iGEM ligation protocol. Then the transformation was carried out with NED-5 alpha cells as used in the previous protocol.

September 13th 2016

All liquid colonies have grown successfully as suspected and there was no growth in the control. Today we carried out a plate reader experiment at 30oC for 20 hours. Using the Cut Smart digest protocol we carried out a digest of the mini-preps. The plate format used can be found here (INSERT PIC OF WELL SAMPLE). We carried out a Gel Electrophoresis as well with the ladder in well 4. We replaced the DNA 2.0 sample with Sample 4. The gel showed that there was NO DNA in samples 5-10. Matt says that there is probably something wrong with the mini-prep.

September 14th 2016

We checked the plate reader in the morning. The gain was too low so the fluorescence was saturated after 8 hours. We tried again with a new plate but the computer broke and we had to wait for IT to come and fix it. We have decided that next time we will use auto-gain function and allow the plate reader to run for 24 hours. In the meantime we also researched Luciferase for new potential constructs.

September 15th 2016

We inoculated fresh LB broth for use with the plate reader. We set up the plate reader again for 30oC that would go up to 42oC where it would stay for an hour.