Question

Observe the given picture carefully. A mixture of DNA with fragments ranging from 100 base pairs to 1800 base pairs were separated by electrophoresis on agarose gel with the following arrangement:

1. What result will be obtained in staining with ethidium bromide? Explain with reasons.     [1]
2. The above setup was modified as shown below and a band with 100 base pairs was obtained at X.
   
   What changes were made to the previous design to get a band at ‘X’. Why did the band appear at ‘X’?     [2]

Answer 1

1. You will get no (or very faint) bands after EtBr staining; the DNA will have migrated out of the gel instead of into it (or will remain as a faint smear near/outside the gel).
   Reason: DNA is negatively charged and migrates toward the positive (anode); in the first drawing, the wells are oriented toward the anode so the fragments move out of the gel. Ethidium bromide only makes whatever DNA remains in the gel fluoresce, so if the DNA is left in the gel, you see nothing.
2. Change made: the gel/electrode arrangement was reversed so the wells are now next to the negative electrode (cathode) and the electric field directs DNA from the wells into the gel toward the positive electrode. Why the 100 bp band appears at X: smaller fragments migrate faster through agarose, so the 100 bp fragment travels farthest from the wells and appears at position X (the furthest/expected position for the smallest fragment). The need to correct electrode orientation is the same reason given above for the original lack of bands.