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 inside cells for freezing by adding 40 % glycerol and LB buffer in 50/50 amounts.
Made a sub-culture of E. coli 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 E. coli Top10 cells then checked that there was enough DNA extracted and estimated its purity using the Nanodrop. A restriction digest of pMS82 was carried out using Kpn1 and HindIII and pAU3-45 using Not1. An agarose gel was made. Agarose gel electrophoresis was then performed on the restriction digest products to check that the correct plasmids had been transformed into the E. coli cells, should get certain fragment sizes - the insert should be roughly 1Kb and the plasmid 6Kb fig 1, The double bands in lanes 3 and 5 show that the insert is the right size.
Poured 12 SFM plates + 1 ml MgCl2 for the conjugation set-up. There are 3 Streptomyces strains taking part in conjugation with E. coli: S. coelicolor (A3) M145 (receiving pAU3-45 with the antGp-neo construct), Streptomyces S4 (receiving pAU3-45 with the antGp-neo construct) and Streptomyces S4 (△antA::apr) (receiving pMS82 with the antGp-neo construct). There will be 4 plates used per conjugation.
Made spore stocks of the 3 Streptomyces strains in preparation for conjugation. Added 50 ul of both S4 strains separately to 2 x yt (yeast extract and tryptone) to make a total volume of 500 ul. Added 5 ul of M145 to 2 x yt to make a total volume of 500 ul. All stocks were incubated at 55 °C for 10 mins.
Added 1.5 ml of 2 x yt to E. coli cells that had been centrifuged (containing plasmids pMS82 and pAU3-45). Aliquots of 500 ul of these cells were mixed with 500 ul of the prepared spore stocks. Dilutions of the conjugations were then made up to 10^-3 and were all plated onto SFM + MgCl2 in addition to the orignal mix (= 4 plates per Strep. strain).
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.