No bands for PCR or ladders

[phant0m]

Hi, I'm using a 4% gel to run 8 samples that vary by buffer and 2 ladders (25 and 100bp) at 130 volts for 30 min.

My question is: what would cause a lack of bands to show up? I've tried twice and I still have nothing. Someone else working in my lab got bands to show, so it cant be EtBd or any other products used in the process. Does anyone know of any common errors that might make a gel not run properly? I'm wondering if I'm making my gel wrong, but it doesn't appear odd.

I'm not expecting anyone to read this and be able to post something like "This is definitely what you're doing wrong..." but I'm an undergrad student and I'd really like to walk in on Tuesday with a good idea of what I may need to change to make my gel work.

[ramnivas]

I think you should use 2% agarose gel made in 1X TBE buffer having proper amount of EtBr in it. I think it will give clear bands in your gel because i m using it daily and getting very good results with 100bp, 50bp ladder. If you still don't clear bands then stain your gel with some amout of EtBr or make fresh gel having more amount of EtBr. and If you are using Genomic DNA then run it on 100V or in case of Plasmid DNA run it on 80V for 1 hour.

[phant0m]

That's my problem, another student got results using the 4% gel at 130 volts. Also, we only stain in EtBr, we don't use it when making the gel.

I'm not so much looking for errors in the numbers chosen, since someone has gotten results with them, as I'm looking for common mistakes made while making or running a gel.

[butters]

from the face that ur ladders also did not show up tell us that there might be problem with the prestain gel preparation. well u can check these

1. When taking gel image make sure to switch on ur UV
2. Have you put correct amount etbr in ur staining tray?
3. wrong orientation for electric current
4. buffer? do you share buffer with other? if yes did their working?

I would suggest you ask ur friend that is getting result to run the samples for you. While you stand beside him and observe what is the difference in ur preparation.

Cheers and do update us.

[lavendergirl]

If you feel confident that you have turned on the UV light, it is likely that you have run the gel in the wrong direction. Remember: the samples 'run to red' meaning that when you turn on the voltage, the samples will move toward the red electrode. If you have the gel backwards, they will run off of the gel in the wrong direction and spill into the buffer, giving you a blank result.

Could also be EtBr - try running your samples with EtBr in the running buffer. Put 1-2 uL in the end of the gel (on the red side) as well as staining it; this will help you visualize the bands!

Good luck and I hope this helped!

[Multiplexion]

Maybe there is DNAse in the electrophoresis buffer due to bacterial contamination. Prepare the buffer anew and try it again.