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Team:UNIK Copenhagen/Signe Notebook
From 2013.igem.org
Notebook
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Week 1 - July 8th-14th
Culturing of magnetotactic bacteria (MTBs)
Making base media for growing magnetotactic bacteria. This media was also used to make agar plates.
MS-1 inoculated in liquid base medium. Falcon tubes were flushed with the nitrogen before use and incubated at 28 degrees.
Plates were kept in a closed container with an Oxoid AnaeroGen pad.
Assembly of constructs
Glycerol stock of E. coli with pBBR1MCS-2 was thawed to grow on these plates.
Primers for MamC (MS-1 strain) was ordered.
Team photo in the park at Frederiksberg Campus
Week 2
Assembly of constructs
Overnight liquid cultures of pBBR1MCS-2 and eGFP plasmid (Adam Takos)
Touchdown PCR amplification of MamC from MS-1 gDNA (both Phusion and Hotmaster) and of eGFP (only Hotmaster).
Gel purification of bands from PCR amplification and subsequent nanodrop. Due to poor yield from this extraction gradient PCR was performed (56-68 degrees).
Traditional cloning of eGFP fragments into pDRIVE.
Transformation of eFbFP-pEX-A (synthesized gene, Eurofins) and eGFP-pDRIVE.
Week 3
Assembly of constructs
Colony PCR of eFbFP-pEX-A and eGFP-pDRIVE. Only eFbFP was succesful.
Overnight cultures were made of colony 1-4 (eFbFP-pEX-A).
Miniprep of eFbFP-pEX-A cultures and nanodrop. Samples sent for sequencing.
Colony PCR of eGFP-pDRIVE was repeated. Still negative.
Cloning and transformation was also repeated. CloneJET PCR cloning kit was used to create eGFP-pJET (pJET was chosen instead of pDRIVE). Not successful.
Gradient PCR of eGFP premade vector (56-65 degrees). Gel electrophoresis was carried out and the bands were purified from the gel.
Cloning of eGFP into pDRIVE. Not successful.
Cloning of eFbFP into expression vector pBBR1MCS-2.
Colony PCR of eGFP-pDRIVE and eFbFP-pBBR1MCS-2. Gel electrophoresis only reveal one colony to be successful (eFbFP-pBBR1MCS-2 no. 17).
Culturing of MTBs
making charcoal media and plates. MS-1 and MSR-1 was inoculated. Liquid cultures were setup in Hungate tubes 200ul, 400ul and 600ul of each strain (MS-1 and MSR-1). Charcoal plates were inoculated as well with both strains.
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