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.
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
- 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
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