It may be that both amplimers are actually the same thing but just started running defferently. Have you changed the size standard recently or the intercalating dye. Some of the modern dna dyes do not work the same pre loaded in the agarose compared with adding in the loading buffer just before sample loading. What fluorescent dye are you using.

Is there any possibility of you picking up the wrong primer set?

Paul Rutland, I have made a new working stock of primers to rule out the possibility of any cross-contamination. In addition, I have used everything fresh like dNTP, enzyme buffer, autoclaved water and even template DNA too. I am using EtBr to visualize DNA. Here I have attached the gel image of both PCR products run on a 1.5%gel.

The electrophoresis runs are inconsistent. The 1st ran much longer than the 2nd and why do you have two ladders on the first and no ladders on the second. Are the ladders the same? Also the 10th band on the right ladder on the 1st PCR picture is the same 10th band on the repeated PCR picture yet one is labeled 400bp and one is 500 bp?

Re-do the TBE buffer have a fresh stock that is the same for both gels.

Make the same percentage 1.5% for both gels.

Run the gels at the same voltage for the exact same amount of time.

Hi Divya Dinesh , Nice picture

what are the dna ladders please....size and company. The left hand side ladders look different to me but that may just be the gel running parameters

Hi, Antonio Ramirez the ladder on the left-hand side of both the gels is of Neb 1kb ladder N3232S and the right-hand side ladder is of 50bp neb N3236S.

I will run the samples again as you have mentioned.

Hi, Paul Rutland thank you for the compliment. I have mentioned the catalogue number of both ladders in the above comment. When you mentioned the dyes, I recalled that in the first gel, I used just 50%glycerol and sample in a 1:1 ratio during loading the samples and in the second gel I used a 1x dye containing bromophenol blue (0.25%w/v) and glycerol (30%v/v) also in a 1:1 ratio.

Now, I repeated the gel run using the samples used in the 2nd image using just 50%glycerol in a similar manner and the band size came down from 500bp to a lower size. Why did this difference happen just because of bromophenol blue I am not able to understand. Do you have any explanation for this?

thank you for the ladder information Divya Dinesh . The patterns looked odd because some of the smaller bands have run off the end of the gel and it was hard identifying the strong bands at 3kb and 500bp. Ithink that the loading dyes have too much glycerol. Typical loading dyes sold as 10x concentration contain 30% glycerol so the final concentartion in your loaded samples should be about 3-5% glycerol but your glycerol concentrations are 25% and 15% on these 2 runs. I do not know if this makes a large difference in running speed but I could imagine that a sample with 25% is quite dense and might slow the diffusion of buffer into the sample which might then move more slowly out of the well compared to the ladder .

It would be interesting to try running a sample with a final percentage of 5% against one of your stronger mixes