PCR Puzzle- Unusal Bands - Molecular Biology

I ran a PCR and then used GEL Electrophoresis to check out my bands. The problem is that that none of my bands came out properly. There is a dense thin band just below the well (barely barely out of the well) for the ladder , and positive control (1st and 2nd well respectively). I see this line much fainter below my other wells. However, it appears that the new staining bath (Ethidium Bromide) was a bit strong so my gel looks a bit bright. Interesting though that the rest of the ladder turned out fine. I am wondering if my template was too dense and contained too much DNA and therefore the DNA is "stuck" in the wells? Why did the DNA from the positive control not travel? I know there was DNA in the template used, because i measured it with a photospectrometer AND ran a gel (which showed decent bands).
My suspicions are: old buffer in gel electrophoresis chamber, bad gel, too much DNA in template.

Anyone have any ideas? or had similar problems? I am very low on DNA extract so i dont want to waste this PCR product.

Thanks

QUOTE (MarineEnthusiast @ Jul 8 2008, 11:45 PM)

I ran a PCR and then used GEL Electrophoresis to check out my bands. The problem is that that none of my bands came out properly. There is a dense thin band just below the well (barely barely out of the well) for the ladder , and positive control (1st and 2nd well respectively). I see this line much fainter below my other wells. However, it appears that the new staining bath (Ethidium Bromide) was a bit strong so my gel looks a bit bright. Interesting though that the rest of the ladder turned out fine. I am wondering if my template was too dense and contained too much DNA and therefore the DNA is "stuck" in the wells? Why did the DNA from the positive control not travel? I know there was DNA in the template used, because i measured it with a photospectrometer AND ran a gel (which showed decent bands).
My suspicions are: old buffer in gel electrophoresis chamber, bad gel, too much DNA in template.

Anyone have any ideas? or had similar problems? I am very low on DNA extract so i dont want to waste this PCR product.

Thanks

This picture might be easier to see the small bands in the first two wells:

Unfortunately you don't tell us anything. (DNA amount, ladder size, sample type, PCR type) So it is kind of hard to answer your question. Near the wells there may be genomic DNA... but in your marker??? The faint bands in the bottom can be your PCR primers (depending on the agarose % and your ladder size) So it is possible that the reaction did not work at all. Who knows?

QUOTE (Kupac @ Jul 10 2008, 10:18 AM)

Unfortunately you don't tell us anything. (DNA amount, ladder size, sample type, PCR type) So it is kind of hard to answer your question. Near the wells there may be genomic DNA... but in your marker??? The faint bands in the bottom can be your PCR primers (depending on the agarose % and your ladder size) So it is possible that the reaction did not work at all. Who knows?

thank you Kupac, I am sick of the questions asked in this forum, people don't know how to think anymore... moreover, they are afraid of asking their colleagues or even their own suprevisor, because they are afraid of showing it's not working sometimes, well, let me tell it's the same for everybody...

THERE SHOULD BE A POST-IT on each board describing how to ask question regarding PCR reactions and DNA extraction RT and stuff, otherwise, we just lose time trying to find out what the person who has the problem hasn't told us... what can I say ?

Marine:
The gel appear that have lots!!of EtBr (next time just wash it in water for 15-30 min), the buffer was use lots of time and is dirty or it has a precipitation and was use like that. About the reaction per se you don't have amplification the bands in the lower part are primers.

Ph3no:
Take it easy, there is no obligation to help if you know the answer and want to help just write it if don't want is ok other person will do it. This is a place to have fun, learn a little bit each day and make some friends.

PCR Puzzle- Unusal Bands - (Jul/08/2008 )