Question

Topic: Sizing DNA fragments via standard curves using horizontal electrophoresis

Between a low quality ladder (DNA marker) and high quality ladder (Kb ladder), which one produces more accurate estimates on a standard curve? Why?

Answer 1

Poor quality ladder or DNA markers are DNA fragments with 100 bp differences (100bp, 500 bp..up to 1Kb). On the other hand, kb ladders or high quality ladders will have DNA fragment with 1 kb differences. DNA markers are preferred for standard curve determination. In a standard curve, the molecular weight of different marker band is noted and log10 values are calculated for each band. The distance travelled by the band in cm is obtained. A XY scatter plot of log10 values (Y-axis) and distance travelled in cm (X-axis) is plotted. The unknown concentration distance is calculated. It is plotted to find the log 10 values. Antilog of it will give DNA in bp.

A 100 kb DNA ladder separates into distinct bands on a gel. If a 1 lb ladder is used, a higher percentage gel needs to be prepared. Further, as the DNA fragments are larger, their migration is hindered within the pores. Hence, separation is not distinct. It is thus, difficult, to calculate the distance migrated.

Thus, for a standard curve quantification, a poor quality ladder (DNA marker of 100 bp ) is preferred.