Hello,
I've been having an issue with my PCR amplicon appearing smaller on a gel than it should be. For reference, the band should be ~500bp, but it consistently runs closer to 250 bp according to the 1kb ladder I'm using. I initially thought my reaction wasn't working, but I sent it off for sequencing and I do have the 500 bp amplicon I was aiming for. So, does anyone know any reasons this could be happening? I'm loading 5 uL of my reaction on a 1% agarose gel, running in 1x TAE. Thank you!
It is possible that your DNA product is forming secondary structures make it more compact and run faster. How fast are you running your gel? Temperature variations can cause irregularities in the pore size of the gel. During electrophoresis, uneven heat distribution can cause samples in the warmer center of the gel to migrate faster than at the samples nearer the cooler edges
David Heisler I'm running it at 100 v, which is the usual voltage I use for most gels. I'm also wondering if it could be secondary structures, I'll look into that. Thank you for the response!
It can be important to vortex the ladder before using it, especially if it is old. Sometimes it does not run properly otherwise. Also, it can actually just get too old and need replacing.
You could try a quick boil and chill of the ladder. Or, try a few different ladders side by side to see if your really is running oddly. Some companies will give you a small free sample too, New England Biolabs often gives out small trial sizes of their DNA ladders
Double-check the sizes of the bands in your DNA ladder. There are many nearly identical versions from each company. Take a look at the stock tube and look up the exact product. I've run into that issue when someone ordered "1KB ladder" and thought it was the same as "1KB+ ladder"
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