Hi
I have run gradient PCR to choose the optimum annealing temperature (picture attached below). In the picture, the amplicons (in red coloured box) obtained at 5 different annealing temperatures look the same. How do I determine which temperature is the best based on the band if all the bands look similar?
Dear Yugenraj,
They do infact appear the same but I would go for the first as it looks a little stronger than the rest. Equally, the bands look slightly different to me by a few bps but that could be because the gel is not straight. Goodluck
the banding patterns are looks same. go for any temperature. i would recommend the highest one.
Thank you all for your responses.
Dear Qin Qi
If you look at the same image in the previous attachment, the bands at the lane 2-6, they all appeared the same. So like your suggestion, I chose annealing temp (54.3 degree C -band at lane no 5). For positive control (which i used for gradient pcr) n for some samples, I get single band at expected size..For other samples, I get multiple bands or single band at different size (attaching new image here). Which is why Im very worried about the optimum annealing temperature . Any insights on this from anyone?
Dear Qin Qi
I'm trying to detect a toxin named, nonhaemolytic enterotoxin (nhe) which has three components nheA, nheB and nheC. I run gradient PCR for all three genes using positive control. In gradient PCR for nheA, I can see differentiate the bands and see at which temperature a clear, bright band is generated. But for nheB and nheC, the bands generated look same for the annealing temperatures tested. So, I chose one of the annealing temperatures to amplify nheB gene, the same temperature that yielded a nice bright band for positive control but resulted in multiple bands or band at different size. Which is why before I do PCR for nheC gene, I wanted to confirm how to choose optimum annealing temperature if all the bands produced at different Ta look similar.
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