I need to use 1 ul of 3-10 ng of my pcr product to sequence by BigDye 3.1 protocol. How can i measure the concentration of my pcr product out of the gel ladder/standard?
Most DNA standards have reference bands with predetermined concentrations of DNA. In some cases all the bands will have the same concentration, while the remainder may be of higher or lower concentration, giving you a range of florescence/concentrations to work with. If you load the prescribed amount of DNA ladder, you can approximate a concentration of your sample by comparing the relative band intensities. As specific size of the fragment may influence fluorescence, as more or less dye intercalates causing more or less fluorescence, try to compare your product with a band in the DNA standard of the same size. Hope that helps.
Hi Miriam,
by gelelectrophoresis you can not measure the dna concentration, but you can estimate it. But in fact this can be quite accurate. I would suggest the following: laod a suitable amount of the DNA ladder (where the mass the band contains is known), load your sample in several dillutions (e.g. 1:100, 1:10, 1 µl...) and, if you are unsecure load half the volume (from lane 1) of the ladder, run your gel, stain and than compare the band intensities of your pcr product) with the band intensities of the ladder. Then you know how many ng are contained in your band and you just have to calculate it according the dillution factor.
hope that helps you,
best,
GAD
But if the intensity of my band is higher than the intensity of ladder at that position, my band at 0.5 which corresponds to 42ng of 1kb ladder from NEB. The intensity of my band same as the intensity of ladder band at 3.0 kb = 125 ng.
that's why it is usefull to load dillutions of your product (see above).
best
GAD
In order to be more accurate, you can use quantity one (a gel doc program that is used to make photos of your gels). This program has the option to measure the density of your bands, hence you can compare the density of a known band (the ladder, as others comment), and the intensity of your own band.
Another option might be to use a nanodrop, or even a spectrophotometer at 260 nm (in this case you need to find the relationship between absorbance and dna concentration, maybe by doing a calibration line).
Hope it helps. good luck
through gel electrophoresis you can generally estimate the approximate concentration of DNA. And it can be quite accurate. I would suggest you to load an appropriate amount of the DNA ladder (where the bands are known), load your sample in several dilutions (e.g. 1:10, 1:20, 1 µl...) and run your the gel, On finish you can use Gel doc or UV transilluminator to compare the band intensities of your DNA ( PCR product) with the band intensities of the ladder. Then you will get to know how many ng are contained in your band and you just have to calculate it according the dilution factor.
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