Grown on 10% HBA + selective supplement dent
DNA extraction (Qiagen)
PCR:
Wild Type + Mutant knockout samples:
-8.5μl milli-q water
-1μl forward primer corresponding to the gene that is knocked out (fecA1, fecA2 & frpB1)
-1μl reverse primer corresponding to the gene that is knocked out (fecA1, fecA2 & frpB1)
-2μl HP DNA
-12.5μl Go Taq green master mix
Negative samples (controls) every second well:
Same minus DNA and 10.5μl milli-q water.
94°C-5 min x 1 cycle
94°C-30 seconds x 35 cycles
55°C-30 seconds x 35 cycles
72°C-30 seconds x 35 cycles
72°C-10 mins x 1 cycle
Run at 100V on 1% agarose gel
How did you do the knockout and what sizes do you expect to see after PCR? The knockout genes still contain binding sites for forward and reverse primer?
First, are you sure that your colonies are H pylori (i.e. do they look like H. pylori?)
If yes, Cornelia is right. The key question is which method did you use to obtain those mutants? Did you perform a gene disruption only or did you do a insertion/deletion? What size do you expect on your gel? assuming you did and insertion/deletion, if the size of the deletion is roughly the same size of the kanamycin cassette then your result (appearing as WT) is expected. If you can, use a primer inside the kanamycin cassette and another one, outside your mutated gene. That way, you should observe an amplification only on your positive mutants.
I've never isolated H. pylori colonies with a random recombination phenotype, the most probable is a contamination. You sure is not letal???
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