Hiya! The fact that your ladder isn't showing up either would suggest that there is a problem with either your gel, your tank or your power pack. Have you tried a different tank? How are you forming your gradient? Does your power pack appear to be working ok and not giving any errors? I struggled for many months with mine and it turned out that my tank was faulty. I went to a different lab and used their equipment and it worked perfectly! Hope this helps!

Hi Lindsay Fulton,

Thank you for the response. I use the gradient forming equipment of Bio-Rad that you need to use the wheel. Sometimes there is an error about rapid change of resistance but after I push the button for continue it did not appear again. I tried to use another powerpac but still the same result. About the gel I prepare it from the protocol published by S. J. Green et al., 2010 ( the formulation is attached in this message). In my next run I am planning to prepare my gel based on the protocol provided by Bio-Ra.

About the tank, how come it can affect the migration. Thank you.

With every details of the steps you have tried out here, probably the power pack didnt work because its obvious that there is no migration here. Most importantly when your DNA ladder didn't even show.

Kindly ensure your tank is fixed properly to the power pack.

Are you using a stacker gel on top of the gradient gel?

The line across the (almost) top of the body of the gel and the absence of a size marker that I know you loaded suggests that the sample has met the transition between the 2 gels and leaked so has run down the outside of the gel not through the gel. If this might be the case then cast the gel in one operation and also make sure that the glycerol is well mixed with the sample and loading dye before loading the sample

Hi Akadiri Olalekan Oladimeji,

Thank you for the reply. I have the same hypothesis regarding the powerpac. But powerpac is sensitive and Usually it will make an error if not properly attached. But I will have an attention to that again.

Thank you.

Hi Paul Rutland ul Rutland,

Thank you for the reply. Yes, I use 0% gradient as top up solution. Your observation could be a possible factor to my problem. I will make sure to apply it next time.

Thank you very much.

Hi all,

Thank you for the suggestions of everybody. Finally the DNA migrated to the gel, but I have new problem regarding the smearing or like degradation of my DNA in the gel. Please if anyone knows or have the same experience with my DGGE result your suggestions and comments are highly appreciated.

The attached picture is the result of my DGGE gel with smearing/ degradation of my DNA in the gel ( 4th and 5 th lanes). The second and 3 rd lanes are ladders ( 1kb and 100 bp) only to check if my DNA has problem.

Thank you

Hi Steve Baeyen,

Thank you for the response. I used only the ladder to check if the ladder can migrate to the gel ( to confirm that I have no problem with my DNA in reference to my first problem). Thank you for the information about the ladder . About to my new problem, thank you with your comments. I will run again tomorrow and I will try to load 300 ng concentration of DNA and different volumes of PCR products to check the difference.

Thank you very much.

I would try to get fresh reagents to overcome smearing. Always helps.