Does agarose go bad? - (Oct/05/2010 )
I'm using Fermentas 1kb GeneRuler now. I thought it was the problem since I used NEB 1kb in my old lab. Then I found a tube of NEB 1kb in my lab. Tried that and.... nope still the same old smear. I wonder if it's the way I set my gel. In my old lab, we pour gels on a glass plate without a cast. Therefore, we had to cool the agarose down till it's viscous enough such that the surface tension would hold the agarose within the edges of the plate and form the thickness of the gel. Now that I use a cast (Bio-rad), I pour it at a higher temperature, maybe 60-70C. The gels need to be thicker too because the combs are fixed-height and higher off the base than I'm used to. I measured it and it's about 4mm. Quite a common thickness I believe, could that be a reason?
I pour gels hot all the time - but if they're thicker than you're used to, are you letting them solidfy all the way? You probably are but it's a suggestion!
I usually let it stand for 1h. Sometime 2h if I have other things to do. But 4mm gels look much thicker than the gels I used to make (maybe 2-3mm), so I wonder if they don't set as evenly. Also, the gels I make now are wider and I found that the combs are 1.5mm thick rather than 1mm at my old lab. How much would the 0.5mm affect the resolution?
Can you post a picture of one of these gels? What is the size (length) of the gel? How long are you running it for? Do the ladders you've tried come in a loading buffer, or are you adding your own? If you're adding your own, what is its composition? Finally, if you're adding your own loading dye, have you run a lane containing just loading dye?
A typical gel looks like the one in the attached pic. My gels used to about 12x8cm and the current one is about 15x7cm (Biorad gel tray). I'm using the Fermentas 6x loading dye now. I'm not sure how we prepared diluted ladder in my old lab but I think we used the provided dye. I'm running it for 20min, same as in my old lab. I'll try running a lane of loading dye next time.
I think the resolution may be due to my stain. I did a google search and found a website that reviewed Gelstar. The smeared ladder in the photo on that page looks similar to mine, apparently due to the marked increase in sensitivity of Gelstar over EB. I tried using less ladder and it seems to help. I think the size of the wells may be another factor but I can live with that for the moment.