Does the size of the DNA fragment appearing in the agarose gel measured in base pairs (bp) correspond to the number of the bases in the sequence of that DNA fragment? For instance if the size of the DNA fragment in the gel is 400 bp, does it mean that there are also exactly 400 number of bases or base pairs when it is sequenced?
Yes, except that you can't resolve DNA with bp resolution on an agarose gel (that is a "400bp" band could realistically be anywhere from 380 to 420bp), especially when bands are overloaded. Also, bear in mind that sequencing runs often don't yield meaningful data immediately adjacent to the sequencing primer (first 20-30 bp or so).
If you want to get sequence data at the 5' and 3' ends of your insert, you need to either sequence inward using an oligo that targets your vector, or design a sequencing oligo that anneals within your insert (not exactly at the ends), and allows you to sequence towards the direction of the insert ends.
Hi Vivian,
In general, if it is agarose gel and your fragment is linear, then yes. If it is polyacrylamide gel then it is not always. DNA curvature, which is an intrinsic property of the sequence being considered, can alter significantly its electrophoretic mobility.
If your plasmid dna is uncut (not linear), hence supercoiled, you can run it on an agarose gel using the supercoiled dna ladder rather than the standard 1kb ladder to get a better estimate of the plasmid size (in bp).
Hi! It will depends also on the sequencing technology (platform, coverage), also on the DNA sequence (e.g. homopolymers). On the other hand, it will depends on the overload of the gel. (If the electrophorized fragment is linear).
Curvature in linear DNA can occur, but is rarely an issue with typical gene cassettes. AT-rich sequences can promote kinks/bends in the DNA and alter the expected migration.
@Vivian- have you found a discrepancy between the sequence length determined by sequencing vs. electrophoretic mobility? If so, what is the size of the difference in bp?
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