IGEM:Cambridge/2008/Notebook/Magnetic Bacteria/2008/07/31

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 * style="background-color: #F2F2F2" align="center"|  |Main project page


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Transformation of TOP10 for MagA and I0500

 * No colonies formed on the Kanamycin agar plates as we failed to transform cells
 * Transformed 50μL competent TOP10 with 1μL MagA plasmid again
 * Transformed 150μL CaCl2 treated TOP10 in LB broth with I0500 plasmid and 50μL of such cells with pUC plasmid as control
 * MagA transformed TOP10 plated on Ampicillin, Chloramphemicol, Tetracyclin and Kanamycin plates in 1/10 dilution to check plasmid resistance
 * pUC control and I0500 TOP10 on Kanamycin plates
 * All plates incubated at 37°C overnight


 * Mag A PCRed with new MAGA programme on the cool black PCR machine :)
 * 10 cycles in touchdown at 47°C
 * 20 cycles with 78°C annealing temperature
 * PCR product run on 1% agarose gel with SyBr Green
 * Lane 3 - 100bp ladder (1μL ladder + 7μL TAE + 2μL dye)
 * Lanes 5 and 7 - PCR-ed MagA (5μL DNA + 15μL SDW + 4μL dye)

Result:
 * Bands at around 1.3kb on lanes 5 and 7 corresponding to the size of MagA
 * Smear of DNA along the two lanes
 * Looked like genomic DNA on gel
 * Suspected having too much DNA on gel and suggested to do single colony PCR

Contact L.E. Bertani at Caltech for MagA gene map!!!


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