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Team:UEA-JIC Norwich/Weekthirteen
From 2011.igem.org
Monday 4th September
Ben Jevans carried out a PCR of the miniprepped Ble and G-Luc plasmids and ran a gel, then performed another mini-prep and gel on twelve previously made cultures. The results of the first mini-prep look good and well as the results from the second mini-prep.
Tuesday 6th September
A restriction digest and gel were performed on the pSAD and G-Luc product, results here looked fairly decent. Mark and Mario return to boot up some more transformation and testing in algae, they get all the equipment ready for the following days to come and do a restriction digest on the G-Luc plasmid so it is linearised . Alistair performed a restriction digest using XbaI and SpeI on his CaMV product but got disappointing results as it looked like the CaMV promoter had not been inserted into the intermediate vector.
Wednesday 7th September
Ben Jevans repeated his digest on the pSAD and G-Luc as well as the addition of a new digest of the cholarmphenicol plasmid containing red fluorescent protein (RFP). These were ran on gels and extracted afterwards; once successfully extracted a DNA ligation was performed. Mario and Mark performed a transformation in algae using the conventional glass bead method in collaboration with starch embedding to help ensure a higher chance of transformants. Alistair performed control digests checking the activity of the enzymes to ensure that they are functioning correctly, and they were found to be working correctly. Likely assumption is that the product was not inserted.
Thursday 8th September
Ben Jevans runs a gel for his product of ligation made on the previous day, results attained are not quite what was wanted. Another PCR was then conducted upon the pSAD terminator and arginine biosynthesis genes. A restriction digest was done upon the G-Luc plasmid and all three products were ran on a gel,extracted and then ligated together. A transformation using the chloramphenicol plasmid ligated with the pSAD terminator was performed also. Mark and Mario conducted a second transformation in algae via the method of electroporation and starch embedding, planning for future tests are to begin.
Friday 9th September
Ben Jevans starts the day by carrying out colony PCR on the colonies picked off the plates made and grown the previous day containing the pSAD promoter in the chloramphenicol plasmid. A gel was ran and extraction of the pSAD-T, ARG-biosynthesis and G-Luc digestion products were cloned into a pGEM-T easy vector. Mario and Mark today performed tests in algae to gather some information about its growing conditions and preferred niche. Alistair performed another cloning experiment with purified PCR product for the CaMV promoter and attempted to ligate it into the pGEM-T easy vector, which was left to ligate overnight.
Saturday 10th Spetember
Alistair performed a transformation of his ligation product and Ben Jevans ligation product. Was left to grow overnight
Sunday 11th September
A miniprep was conducted upon the cultures made from colony PCR, this includes E.coli transformed with the ligation product of insert plus the chloramphenicol plasmid and E.coli transformed with ligation product of insert and the pGEM-T easy vector.
