I get quite sharp bands of my amplicon, when I loaded some of it to check the PCR product. But when I electrophoresed the samples all together to elute them, although I got my band but it appeared as a smear from the well.
1. I electrophoresed at 70V. Should I increase or decrease the running voltage?
2. Is it because of higher amount of sample?
3. Or is it because of issues with gel polymerization and EtBr?
The problem could arise from the proteins (i.e polymerase or other proteins such as endonucleases in case you do a colony pcr) in your sample. Pol can still attach to some of your DNA and if you have other protein contaminats, you could have lots of smears infact. I think when you load less simply you dilute the contaminants and get sharp band. But with higher loading smears appear as the concentration of contaminants are higher in this case. So i would recommend to use 0.01 percent to 0.05 percent SDS in your loading dye or treat your sample with this low level of SDS. I hope this will help to get the problem solved.
The problem could arise from the proteins (i.e polymerase or other proteins such as endonucleases in case you do a colony pcr) in your sample. Pol can still attach to some of your DNA and if you have other protein contaminats, you could have lots of smears infact. I think when you load less simply you dilute the contaminants and get sharp band. But with higher loading smears appear as the concentration of contaminants are higher in this case. So i would recommend to use 0.01 percent to 0.05 percent SDS in your loading dye or treat your sample with this low level of SDS. I hope this will help to get the problem solved.
I think that the voltage is Ok because the first sample ran well. More likely is that the gel was used too soon (or without buffer in the gel )and was not completely set so when the voltage was turned on the heating effect softened it too much. If you used 1M betaine or a high salt pcr buffer and loaded large samples then there might be some non uniformity in the gel running due to high salt but I would expect that a well set agarose at 0.8% or higher and fresh buffer should run well. If your laboratory is very hot then allow the gel to set then put it in a fridge for a few minutes prior to loading .Do not set it from liquid in the fridge or you get uneven setting and surface patterns on the gel. Check that the current is similar to previous runs in case someone has changed the running buffer concentration or constitution
It also depends on what your sample is. You haven't mentioned whether your sample is nuclic acid or Protein. In case of DNA if you get smears near the well that means some secondary structure is present in your sample. Or it might also come if you run single stranded DNA or sticky ended dsDNA. In thay case you may decrease the temperature of running to 40C or something.
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