Hi Najib,

You might need to optimise your MgCl2 concentration. Do you know what your annealing temperature for your PCR is? Incompatible annealing temperature to the melting temperature of your primers might cause the smearing. It could be that your DNA is degraded as well. So there are a few different possibilities. What have you troubleshoot so far?

Kind regards,

Dian

Dear Najib,

how about the DNA ladder?

Is it also smeary? When we had a problem with this, our milliq water was in bad quality, full of endotoxins and whatever.

We simply bought destilled water to make our TAE and TBA stocks and running buffer and the problem dissapeared.

Best Regards,

Ferenc

thank you all...I will  rectify the source of problem soon and come back to you later.

if you are using a high fidelity polymerase reduce the amount of polymerase. as some time these enzymes degrade the primers and that is the main cause of smeary gel. for example if you are using 0.5ul polymerase in 50ul pcr reaction reduced it to 0.25  or so on. hope it will work.

Can you please show a problematic gel with a correct size marker?

1. Proteins got degraded. Need to check protein concentration if needed before loading. Save the gels after transfer and stain them with Coomassie blue stain to check if there are still some proteins left and did not transfer properly.

2. The gels expired (in case you are using pre-cast gels).

3. Overload of samples.

4. Change in the voltage while running.

Check if when gel is heating during electrophoresis. If so, decrease the voltage and smeary bands may be avoided. Especially if DNA marker is also smeary that is an indication that problem is in the electrophoresis step.

Dear Najib,

It can be because of DNA not being purified properly (check with Nanodrop) or degraded. It can also be due to PCR problems. Try to optimize your primer pair with 'thermal gradient PCR´, if not done already. Try making fresh gel and buffer. Optimizing the running voltage is another thing to consider.

Good luck 

I agree with Nikolay: running your gel too fast can cause the bands to blur.  However if this is a sudden new problem, reagents might be the problem. We had a similar problem when our reverse osmosis water purification filter failed and the water quality dropped without us realising - lots of the gels ran poorly for a while.  Another easy mistake is to make a gel with TAE and put it in a tank where someone has been using TBE....  make everything fresh and give it another go.

Good luck.

Thank you all for your suggestions and opinions.