Without knowing exactly what you are amplifying, and assuming that the bands halfway down the top portion of your gel are your desired product, I would assume you have severe mispriming. The lower bright bands look like primer dimer, or some small product. They are heavily present in all but one lane. Once cycling starts, kinetically, the smaller PCR products will amplify faster than the larger product. My suggestion is to start by increasing your annealing temperature. If that doesn't work, then I suggest redesigning your primers.

Hi Mariana,

I completly agree that with David Burden that the lower bands in your gel are very likely due to the formation of primer dimers. They might be also a small product forming.

However, I also see a double band if I look at your specific band - is this intended?

Running a temperature gradient for your primers is always a good idea if you have difficulties.

You might also consider new primers but is seems to me that you are doing genotyping and sometimes it is not so easy to use other primer sequences.

However there are some additional points/Ideas

What I am missing on your gel is a negative/no template control - this could show if the lower bands are due to primer dimers ( even if you have one lane wit no bands this does not autmatically exclude the posibility).

• so please do include a negative/no template control

I also see no positive control. You should include a positive control you know that is working to see if you have problems with your template or other components of your PCR reaction.

• so please include a positive control

I don't know what is your template - I assume it is genomic DNA as this seems to be a genotyping reaction.

If you do gentotyping and you get a great variation in your results this might be due to differences in the template quality, so it might help if you try another method of DNA isolation.

I also would not recommend to use to much of your template because sometimes you can have a lot of inhibitors (or increase your PCR volume -15 µl is a reltive smali volume) - this depends strongly on your isolation method.

Sometimes you also have to try another polymerase to get good results - from my experience people tend to use cheaper polymerases for genotyping because they often do a lot of reactions and most of the time this works. However, sometimes you have to get a High fidelity polymerase or a polymerase which gets good result with CG-rich sequences.

In addition, if you have difficulties a would always use a Hot start polymerase (if you are not doing so already) or at least to perform a manual hot start.

Here is what I would do first ( I myself did a lot of mouse genotyping but I think this would also work with other organisms)

Get a high quality genomic DNA from the organism you are working with. (here you might use another protocol as for genotyping because I now that you often use fast and more convient protocols during genotyping).

Establish your PCR with the high quality DNA (control your template DNA quality on a gel) to determine:

• optimal annealing temperature - run a gradient if possible
• Optimal Annealing time (prolonged times cann facilitate the formation of primer dimers and mispriming - you might try 15 sec)
• working polymerase (hot start might be helpful)
• amount of template (don't use to much - for genomic DNA 50 ng should be enough)
• How many cycles you need for good amplifikation
• establish positive and negative controls (if you don't have them already)

If you can't establish a working PCR at this point you might consider new primers if possible

If you get good PCR results then you can use the established protocol for genotyping and if you still have difficulties you should reconsider using annother method of DNA isolation.

The rest of your protocol sound reasonable to me (nevertheless also modification here can improve outcome like decreasing your annealing time)

And just one final thing - even if it is surely not the main point here. A 2% gel is a high concentration for a gel - I don't now the size of your expected product - however as you have an extension time of 50 sec I assume it is not a really small product and I would reduce the agar concentration to 1 % (than it might be also easier to see if your upper band is only one band or if there are two bands).

I know this is a lot but I hope it might help - if you have question feel free to ask

KR Dörthe

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