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PCR analysis - why are DNA fragments different sizes from markers?

PCR analysis - why are DNA fragments different sizes from markers? - PCR - Polymerase Chain Reaction Forum

PCR analysis - why are DNA fragments different sizes from markers? - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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Old 08-06-2011, 06:16 PM
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Default PCR analysis - why are DNA fragments different sizes from markers?



Have calculated the marker size in base pairs and plotted on a calibration curve. I then measured relative mobility of the DNA fragments and from my graph have obtained their base pairs. However, when I compared the DNA frags to markers on my gel, the base pairs of the DNA do not match up with where the markers are, they are higher up than some of the markers and yet have a lower base pair number. Could this be because my line of best fit on my calibration curve is wrong, or could there be some other reason? Please help!
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Old 08-08-2011, 01:21 AM
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Default Re: PCR analysis - why are DNA fragments different sizes from markers?

sometimes overloading of the marker/ high marker concentration will give a thicker band that might affect your calibration curve, same for DNA. If the fragment is a PCR amplicon, then its best you sequence the fragment as it may be non-specific amplification of product. if the fragment is plasmid DNA, then u can expect 3 bands of different sizes- linear, supercoiled and nicked-circular; each will migrate differently.
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