IGEM:University of East Anglia (UEA), Norwich, UK/2009/Notebook/NRP-UEA-Norwich iGEM/2013/07/26: Difference between revisions
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==Floor Two== | ==Floor Two== | ||
The transformation reactions from the previous day were retrieved and | The transformation reactions from the previous day were retrieved ''Fig.2,'' Colonies had grown on both plates and were red. Colony Polymerase Chain Reactions (PCR) were preformed and the results were analysed by gel electrophoresis ''Fig.3.'' | ||
[[Image:WP 000051.jpg|thumb|Fig 2:Two LB agar plates with Chlorophenicol antibiotic containing BBa_J04450 and J04450 with added NdeI site respectively.]] | |||
As the western blot from the previous day was unsuccessful, the protein analysis took a different path. The soluble protein fractions were purified using a His Mag solution to select the His Tagged AntA σ factor. These samples were ran on a 15% SDS PAGE along side the unpurified soluble fractions for 1 hour 25 minutes and stained with instant blue dye. | As the western blot from the previous day was unsuccessful, the protein analysis took a different path. The soluble protein fractions were purified using a His Mag solution to select the His Tagged AntA σ factor. These samples were ran on a 15% SDS PAGE along side the unpurified soluble fractions for 1 hour 25 minutes and stained with instant blue dye. | ||
[[Image:26 july.JPG|thumb|Fig. Analysis of colony PCR reactions. Lane 1 shows the negative control with no DNA, Lanes 2 and 3 are the positive controls with the original J04450 plasmid and J04450-Nde1 biobrick respectively. Lanes 4 and 5 are the J04450 colonies 1 and 2 respectively. Lanes 6 and 7 are the J04450- Nde1 biobrick colonies 1 and 2 respectively. Lanes 8 and 9 are the ligation reactions sample 1 colonies 1 and 2 respectively, while lanes 10 and 11 are are the ligation reactions sample 2 colonies 1 and 2 respectively]] | [[Image:26 july.JPG|thumb|Fig 3. Analysis of colony PCR reactions. Lane 1 shows the negative control with no DNA, Lanes 2 and 3 are the positive controls with the original J04450 plasmid and J04450-Nde1 biobrick respectively. Lanes 4 and 5 are the J04450 colonies 1 and 2 respectively. Lanes 6 and 7 are the J04450- Nde1 biobrick colonies 1 and 2 respectively. Lanes 8 and 9 are the ligation reactions sample 1 colonies 1 and 2 respectively, while lanes 10 and 11 are are the ligation reactions sample 2 colonies 1 and 2 respectively]] | ||
==Outreach== | ==Outreach== | ||
Revision as of 07:50, 22 August 2013
 Week 8
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Floor OneMade up 6 x 500 ml SFM + Nystatin. Streaked lawns of isolated single Streptomyces colonies that were sporulating. Set up a Polymerase Chain Reaction (PCR) using a toothpick to scrape clear large colonies from 8 selected plates - this acted as template DNA. The Master Mix contained 18 ul Buffer (10 x NH4), 9 ul DMSO (100 %), 3.6 ul dNTPs (10mM), 9 ul Primer 1 (20 uM), 9 ul Primer 2 (20 uM), 5.4 ul MgCl2 (50 mM), 121.5 ul dH20 and 1.8 ul Taq polymerase. A 20 ul aliquot of Master Mix was then added to each of the 8 samples. Floor TwoThe transformation reactions from the previous day were retrieved Fig.2, Colonies had grown on both plates and were red. Colony Polymerase Chain Reactions (PCR) were preformed and the results were analysed by gel electrophoresis Fig.3.  As the western blot from the previous day was unsuccessful, the protein analysis took a different path. The soluble protein fractions were purified using a His Mag solution to select the His Tagged AntA σ factor. These samples were ran on a 15% SDS PAGE along side the unpurified soluble fractions for 1 hour 25 minutes and stained with instant blue dye.  OutreachSome members of the team attended an afternoon for summer students organised by the school of biological sciences at UEA. We spoke about iGEM and our project to other students and members of faculty. Have been in contact with local Defra rep about obtaining a soil licence. Gathered all the correct forms and have handed them over to Matt Hutchings. I have had to put some sediment requests to the US on hold until the licence application is underway. A date has been set for the Forum Event (28th August). Have been designing the advert + discussing what we will do for the event. Also we have identified local printing companies for posters/maps. Also, I have arranged a meeting with Anne Osbourne at the JIC to open a discussion about how iGEM parts can be realistically integrated into real research projects. We are interested to see if our tool-based design could offer a realistic basis for real synthetic biology use in professional research institutions. Perhaps our project is more realistic than other iGEM projects. Will need to devise some questions for Anne before next Tuesday. Does this count as Outreach?? Still interesting if not. Forum Advert Outreach Registry - contact other teams, write the message. Questions for Pro research labs
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