IGEM:University of East Anglia (UEA), Norwich, UK/2009/Notebook/NRP-UEA-Norwich iGEM/2013/07/24
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Prepared pMS82 and pAU3-45 plasmids for freezing by adding 20 % glycerol and LB buffer.
Made a sub-culture of ETpuz cells by adding 300 ul of cultured cells to 10 ml of LB broth with 10 ul of the appropriate antibiotics (Apr, Hyg and Chl).
Performed a Qiagen DNA extraction (mini prep) of Top10 cells then carried out a restriction digest of pMS82 using Kpn1 and HindIII and pAU3-45 using Not1.
The samples 2b2 and 3b1 were digested with restriction enzymes Nde1 and Xba1 and analysed by gel electrophoresis. Six fragments were cut from the gel: a large and small from each sample (2b2S,2b2L,3b1S,3b1L). These were then purified and ligated to give two samples;one (2b2S and 3b1L) and two (3b1S and 2b2L).
Another two SDS-PAGE's were prepared, each with 12% Polyacrylamide to give a better definition between the bands on the gel. One was stained with instant blue and the other was to be used in further analysis using the western blot technique. The gel was run, on a nitrocellulose membrane, for 1 and a half hours at 55mA using western blot electrophoresis. It was then left on a shaker at 4°C overnight in 10mL blocking solution.
A Forum date for Wednesday the 28th August has been set. All Saturdays were booked right until the end of the year! Booking forms will have to be filled in.
As part of the event we will put together a general iGEM leaflet, which we will also propose to other UK teams to be a part of the 'Outreach database' discussed in earlier posts.
We are in the process of finding out the UEA FedX account number so that the postage costs for soil/sediment samples can be covered.