Hi Ponsekaran,

A picture will tell the true story, but let me do a little bit of guess work for you.

The Golden Rule: Please refer to Gene Cloning by Sambrook & Russel - Volume 1, Chapter 5. Protocol 1. It has all the information for tweaking an agarose gel.

There are a few major reasons why you should see a wavy or twisted bands in agarose gel.

• Non-uniform solidification of the agarose gel / impurities in the gel - I have experienced that when the agarose gel is disturbed during the process of solidification it leads to non uniform density along the gel. As a result the bands may appear curved, wavy, or twisted.  if nothing else, please make sure that you have completely dissolved the gel before you cast it.
• Changes in direction of the electric field - if the platinum wire that runs through the anode or cathode is broken the direction of the electric field changes a little bit and tend to put the bands off the straight road.
• Selection of percentage agarose gel: Please use the table I have attached, which I have adopted from Sambrook.
• One stupid stuff I have done in the past: I have made the agarose dissolve in pure milli-q water once, instead of buffer and ran the gel without realizing it. My DNA was running all around.
• The major problem could be your buffer: Some buffers (e.g. TAE) have low buffering capacity and sensitive to high temperature. That buffer is not suited for long running and one of the reason of smiley bands.

Voltage Applied: I will tell you what I do and may be it will help you. I run agarose gels at around 4-5 V/cm. You should calculate the distance between the electrodes for this approach. Please DO NOT use the length of the gel. For example, if the length between the electrodes is 15 cm, I will use 15 * 5 = 75 V for my run. The length of the gel that I run is usually 8 cm and it gives brilliantly resolved bands when using a 1 Kb Fermentos DNA Marker. Please note that when you are running agarose gels at 4-5V/cm you have to run it for around one and a half hour, but in my experience patience pays. I have produced DNA marker bands just like in their instruction manual.

Kind regards

The ladder is made up in low salt buffer and although there is a lot of dna in the ladder it is separated into many bands each of low dna concentration, The smiling effect is usually caused by overloading ( run a smaller sample) or too much salt. Try running the gel at a lower voltage as the larger bands of the ladder do show slight smiling