IGEM:Cambridge/2008/Notebook/Magnetic Bacteria/2008/08/04

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MagA Colonies and PCR

 * Picked another 4 colonies, labelled no.6-9, from the MagA TOP10 plate with Ampicillin
 * PCR such colonies using the MAGA programme on cool black PCR machine
 * Picked 2 single colonies from MagA colony 4 agar plate prepared before with Ampicillin, labelled as 4a and 4b
 * PCR along with the 4 picked colonies
 * MAGA programme with 24 cycles instead of 20

Gel Electrophoresis
PCR Products and 2 I0500 plasmid samples prepared run of 0.8% agarose gel with EtBr on 90V. Lanes of Gel from L to R:


 * Lane 2 - Hyperladder I (5μL DNA)
 * Lane 3 - wx (Voltage)
 * Lane 4 - vw (Voltage)
 * Lane 5 - MagA colony 4a
 * Lane 6 - MagA colony 4b
 * Lane 7 - MagA colony 6
 * Lane 8 - MagA colony 7
 * Lane 9 - MagA colony 8
 * Lane 10 - MagA colony 9
 * Lane 11 - I0500 (1.1μg/mL DNA detected by nanodrop)
 * Lane 12 - I0500 (3.9μg/mL DNA detected by nanodrop)
 * Lane 13 - Supercoiled ladder for I0500 plasmid (5μL DNA)

Result of Gel:
 * Bands of around 1400-1500bp corresponding to MagA present on lanes 5 to 10
 * Bright bands for colony 4b, 6, 7 and are cut out from gel
 * No bands for lanes 11 and 12 for I0500 plasmids
 * TOP10 not transformed with I0500

Re-transformation with I0500 to Competent TOP10

 * Checked DNA concentration of extracted I0500 biobrick from filter paper with nanodrop spectrometer
 * 14.8μg/mL DNA present but a considerable amount of proteins also present in extract which may affect the reading
 * Transform competent TOP10 with I0500 again following standard protocol but using only 40μL TOP10 for I0500 and 10μL TOP10 for pUC control
 * Plated on Kanamycin agar plates after 2 hours of incubation according to protocol

Extraction of Yeast GAL1 promoter BBa_J63006

 * Possible transformation of eukaryotic yeast cells with MagA to see if spontaneous magnetite can be formed as in mammalian cells
 * Extracted biobrick DNA from Plate 1003 Well 2H following standard protocol
 * Transform CaCl2 treated TOP10 cells following standard protocol except that 600μL SOC is added with overnight incubation at room temperature