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

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MagA Recovery

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.

Gel showing bright bands at around 1.4kb on lanes 5-10 corresponding to MagA

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