I have conducted PCR on DNA extracted with boiling method for Pseudomonas aeruginosa and some of the samples had multiple bands. Also, in the same manner i have encountered the same problem with Klebsiella pneumoniae isolates in the detection of another gene. Moreover, I can't detect the exact problem?
Can anyone kindly provide guidance ?
It is possible the colony you picked for the DNA extraction was not single. Bacterial colony unlike fungi, the colony may look the same in terms of colour, shape but different strains. You have to make sure you pick a single colony from a straight line before placing it onto LB broth for incubation.
Also, regularly change the TAE buffer water for the gel electrophoresis.
There can be multiple reasons to this. Majorly could be some contamination, where the primer is binding non specifically. Try repeating the extraction from pure colonies.
This happens when you work with impure isolates (multiple strain). The way forward is to re-purify the isolates and repeat the extraction process.
If you are sure about your extraction and there is no contamination, it could be possible non specific band due to improper annealing so you can increase the annealing temperature or use gradient temperature ,you can also use DMSO to improve specificities of PCR priming reactions.
I will address p. aeruginosa in my answer. Most probably, these samples are considered negatives, meaning that they aren't pseudomonas aeruginosa, thus you can see the non-specific binding that occurred in the rxn. If your goal is only screening for isolating this bacterium, then you shouldn't worry much. On the other hand if you are sure that all are p. aeruginosa, then try optimising the annealing T by conducting a gradient PCR rxn. Do so by looking at the Tms (Melting temperatures) of the F and R primers and choose the one with the lowest Tm, minus it 5 degrees in order to obtain the range you need for the gradient; starting with the -5 T.
- Badges
- Science method