I have a genotyping PCR that results in 2 DNA fragments of 780 bp and 830 bp. I can't change the primers. I have tried a 0.9% gel and thin combs but I still have trouble separating the bands. Does anyone have an idea how to separate these?
You just need to try higher percentage agarose (up to 2%) and run the gel for as long as possible before the fragments fall off the bottom of the gel. If that doesn't work you could try polyacrylamide gel electrophoresis.
You just need to try higher percentage agarose (up to 2%) and run the gel for as long as possible before the fragments fall off the bottom of the gel. If that doesn't work you could try polyacrylamide gel electrophoresis.
As Daniel says, higher % agarose gels (I'd also recommend 2%). You shouldn't even need to run it that long to distinguish a 50bp difference.
It might be helpful to run reference samples containing both DNA fragments amongst your test samples: it's a lot easier to decide if a band is "high" or "low" when you have both bands right next to it for comparison.
Doesn't directly answer your question but for future reference, you may find the following details helpful:
https://www.promega.co.uk/resources/pubhub/enotes/what-percentage-agarose-is-needed-to-sufficiently-resolve-my-dna-sample/
Daniel is right, you need to increase the agarose concentration. I frequently use 2% agarose gels and run them for 1.5 hours to resolve bands of those sizes and smaller.
You can also try a restriction digest that will cut only one of the fragments at a predicted site. This will make it smaller and easier to separate.
I would add you should try with a comb with broader (and thin, as you said) wells.
Like every one else suggested, using a 2% agarose gel is best. However, I would suggest using Sodium Borate Buffer to run the gel. Here is the reference - http://www.biotechniques.com/BiotechniquesJournal/2004/October/Ultra-fast-high-resolution-agarose-electrophoresis-of-DNA-and-RNA-using-low-molarity-conductive-media/biotechniques-117338.html?pageNum=2
Because they are so close in size, I suggest using polyacrylamide gel 12% instead of agarose. & use more glycerol/sucrose than usual to keep the bands thin as possible.
A simple suggestion is to increase the % of agaorose to 2-3% and run the electrophoresis for a longer time with low voltage (e.g. 40 Volts). The bands tend to seperate when run more slowly due to low voltage.
I wish you luck!
Alternatively you apply the following approach:
Perlman D, Chikarmane H Halvorson HO: Improved resolution of DNA fragments in polysaccharide-supplemented agarose gels. Anal Biochem. 163 (1987) 247-254.
Lower the voltage, increase the time, tease the bands out for greater separation. If that doesn't work, use a stronger gel..
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