Getting MagA Out
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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Magnetic Bacteria
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