I'm trying to detect hblA gene by using the PCR method as described by the journal"Enterotoxigenic Profiles of Food-Poisoning and Food-Borne Bacillus cereus Strains".
When I run gel electrophoresis, two bands (2nd well from left) are generated on the gel. One band has the size similar to that of desired fragment which is 1154 bp (according to journal) and the other has smaller size. The image of gel is attached below
Is this because of non specific amplification? Usually non-specific amplification results in vague multiple bands which occur close to each other in a continuous pattern. On the contrary, the two bands I obtained appear away from each other (not continuous) and look distinctive and sharp. Can non specific amplification produce such bands during gel electrophoresis ? Or could there be any other reasons
non specific bands are simply where the primers anneal elsewhere than where you want them to. Amplification can be excellent and clean. There may be a pseudogene or similar sequence other gene or just bad luck and the primers anneal randomly byt close to each other...the genome is huge and there are many places to anneal especially if the annealing temperature of the pcr is too low
In my opinion, the primers used in your experiments are maybe not specific (only for your region of interest). Maybe it is quite similar to other corresponding regions in the DNA used as template in your setting. Then you should identify a more adequate and/or more specific primer oligo.
Another possibility is unspecific binding to your template DNA when the Annealing temperature is too low (maybe you try the same setting with an optimized protocoll.
You better perform a preparetive PCR from your reaction, cut the two bands separately from the gel, gel purify, and send them for sequencing. After receiving the sequences you can judge what is happening based on scientific evidence.
Have you confirmed your primers using insilico tools like ucsc, OK I go calc ?
If they are binding on single site at there then this might be issue related to annealing temp. You must run gradient pcr, if getting same bands then go for sequencing
Thank you for the responses.
I will try to run gradient PCR and also cut the desired band and send it for sequencing.
I would sequence both bands, since both bands are so distinctive and clear. You might discover something new.
Further purification of your gel band will yield desired result. All the best.
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