Question

Lane 2 of a PCR shows no bands, indicating an unsuccessful PCR reaction. The reaction also shows poorly separated bands (larger bands) in the reference ladder lane. Describe whether or not there is a connection between the success of a PCR reaction and how well separated the bands are in your reference ladder.

Answer 1

The ladder showing poorly separated larger bands will indicate a improperly prepared agarose gel. Usually if the gel does not solidify properly, it will cause the protein molecules to separate poorly. Further, there is overloading of ladder will also cause poor separation. If the ladder is more viscous, the separation will be poor. The smaller bands will not be affected much due to their small size making them easy to pass through the pores of the gel.

The PCR land did not show any band. This is possible due to a faulty PCR reaction. If the reaction was successful, then the bands would have appeared fussy in a improperly prepared gel. As there were no bands, this indicates the PCR was unsuccessful. The success of a PCR reaction does not affect the reference ladder, as the reference ladder is not subjected to PCR. The reference ladder is just a series of DNA molecules of different molecular weight that are labeled with different colors (in case of pre-stained ladders). The reference ladder is run along with the samples on the gel to find the molecular weight of the PCR product.