IGEM:University of East Anglia (UEA), Norwich, UK/2009/Notebook/NRP-UEA-Norwich iGEM/2013/08/07: Difference between revisions

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==Floor One==
Gels were viewed under UV light (using the GelDoc). We were able to confirm that the PCR was successful as the primer dimer bands (approx. 100 bp) were visible at the bottom.
Diluted the new soil samples (61-76) from 10^-1 to 10^-4. Plated the dilutions 10^-2 to 10^-4 on SFM + Ny.
Streak purified all remaining contaminated samples.
Lawned single Streptomyces colony plates.
A challenge presented itself when we discovered that the neo gene (for kanamycin resistance) may need to be changed as the reporter gene. Streptomyces sp. (S4 for certain) express AntA transiently, only in one stage of growth (substrate mycelia growth after sporulation but not in aerial hyphae) and so does not harbour kan. resistance after that leading to the death of bacteria. There was talk of using RFP (red fluorescent protein) or GUS (β-glucoronidase) as alternative reporter genes.
==Floor Two==
==Floor Two==
A 15% SDS PAGE was run using the soluble and his-tag purified samples from the protein overexpression at 150V for 1 hour.
A 15% SDS PAGE was run using the soluble and his-tag purified samples from the protein overexpression at 150V for 1 hour.

Revision as of 02:31, 16 August 2013

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Floor One

Gels were viewed under UV light (using the GelDoc). We were able to confirm that the PCR was successful as the primer dimer bands (approx. 100 bp) were visible at the bottom.

Diluted the new soil samples (61-76) from 10^-1 to 10^-4. Plated the dilutions 10^-2 to 10^-4 on SFM + Ny.

Streak purified all remaining contaminated samples.

Lawned single Streptomyces colony plates.

A challenge presented itself when we discovered that the neo gene (for kanamycin resistance) may need to be changed as the reporter gene. Streptomyces sp. (S4 for certain) express AntA transiently, only in one stage of growth (substrate mycelia growth after sporulation but not in aerial hyphae) and so does not harbour kan. resistance after that leading to the death of bacteria. There was talk of using RFP (red fluorescent protein) or GUS (β-glucoronidase) as alternative reporter genes.

Floor Two

A 15% SDS PAGE was run using the soluble and his-tag purified samples from the protein overexpression at 150V for 1 hour.

Outreach

The flights for the European Jamboree was booked.



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