Saugat, GelRed does affect the migration of the DNA

You are probably using too much stain. What I do is to use 1 uL of stain into 50 or 100 mL gel (there is no difference in either volume of the gel), load the sample and run them. You can watch the gel right away. So, definitely either 1 uL in 5 or in 50 uL, is way too much stain.

On the other hand, in the Biotium web page they day: "To avoid any interference the dye may have on DNA migration, we recommend using the post-staining protocol. If your application requires loading more than the recommended amount of DNA, use the post-staining protocol". Thus, if it is important to know the precise size of the fragment, I would use their recommendation, which means, making the gel without stain, running samples also without stain and after running the gel, staining the gel. The manufacturer recommends adding 30 uL of stain in 100 mL of distilled water and staining for around 30 min. If there is too much background (which will depend on the non-specific amplification and/or excess of initial DNA), then destain in water for few minutes.

I hope this helps

I also use gel-red and never faced such problem. I dilute gel red in 1:10 ratio in distilled water and keep it as working gel red solution. For 50ml gel I add just 5ul of working gel red solution. I also think your problem is you are using too concentrated gelred. Hope it helps. 

I agree with previous answers. Gel Red carries two positives charges. As you increased its amount, you decrease the DNA charge, so , at a given time  the change of DNA mobility apperas, enhanced by the fact that the DNA quantity in your PCR sample is not the same as in the ruler.

Dear  Sougat,

As I read in some answers, you are using GelRed in high concentration. In our laboratory, we dilute 0.6 ul of GelRed 10.000X in 1 ml of generuler 6X and it works perfectly.

But you are right about the migration rate modification when using GelRed, in fact, suppliers warns you about this in the the product information sheet (http://biotium.com/wp-content/uploads/2013/07/PI-41003.pdf), look at Staining Protocols section. I had some problems and asked directly to the company, here is part of our e-mail conversation:

"According to the information, that we have received the problem most probably is caused by mixing DNA ladder with GelRed dye before electrophoresis. GelRed is a nucleic acid binding dye, thus staining prior to electrophoresis affects DNA migration. Also it depends on how much of DNA is loaded in gel. The more DNA is loaded, the more GelRed dye binds to DNA and distort migration of DNA fragments.

We strongly recommend to stain the gel only after electrophoresis with GelRed and to follow Post Gel Staining protocol provided by the GelRed suppliers (http://biotium.com/wp-content/uploads/2013/07/PI-41003.pdf). It should help to solve the problem".

I hope you find this information useful.

Kind regards

Let do it as simple way: You need to dilute your PCR product. The density of your PCR product should be the same of the band from your DNA ladder. For instance, You can load 2 ul of PCR product and 2ul of 1/30 diluted of your PCR product. You will see the different. Good luck