Does agarose go bad? - (Oct/05/2010 )
I've started work in a new lab for a month or so now but I've not been getting good resolution of bands >3kb in the DNA ladder. The lower bands are defined but those above 3kb are thick and smear. I was using TAE and tried 90V-120V. Higher voltage seemed to improve the resolution but still nothing as good as what I used to get in my last lab. Using new TAE didn't improve the resolution. I thought it could be the buffer, since I used SB in my last lab. However, I still don't get good resolution. I tried new DNA ladder, still no difference. I'm now wondering if the agarose is old (based on the batch no., it's 2007) and has degraded or changed in some ways (absorption of moisture?) detrimental to resolution. How long is the shelf life of agarose? Does old agarose give poorer resolution?
I do have one agarose, still using from 2007 and is still works fine.
Perhaps you should try 0.5X TBE for your gel and running buffer.
what concentration of agarose are you using? if the concentration is too high, you wont get a good resolution for the large bands, if too low no resolution for the smaller ones. usually 1xTAE with 80 V should produce good results for a minigel.
Have you tried to use less of the marker? Usually we use half the recommeded concentration and get perfect results (you just need to be aware of the dilution when doing quantifications).
I've already tried TAE and SB but both gave suboptimal resolution, so I'm suspecting something other than buffer. I'm using 0.8% agarose. It's on the low side so if any problem should arise, it should be resolving the smaller DNA but I have problem with the larger ones. I'm using 1/5 the recommended concentration of DNA ladder actually (100 instead of 500ug).
I've done these for almost 5 years at my last lab with no problem. Other changes are (1) using Gelstar here instead of ethidium bromide (2) casting gels in a mold instead of on a glass plate (yes, ONLY glass plate, relying solely on surface tension to form the gel thickness) (3) different power supply.
Are you using another brand of agarose than in your previous lab? there are huge differences here (also with the cheap agarose). No experience with Gelstar, but could be a reason....do you have the possibilty to try EtBr on your gel somewhere?
It's the same brand of agarose (Sigma, for routine use) but a smaller packaging. Too much hassle trying to use EtBr at my place (bureaucracy), so maybe I'll try post-staining and see if there's any difference.
is it the same lot that you used before. there may be lot to lot variation (personally, i think the difference may be due to storage conditions).
The gel chamber might also have an influence, some chambers are "better" than others (perhaps a design problem of some manufacturers). And of course the length of the chamber. The longer the migration distance, the better the separation.
Agarose: Only same brand, or also same type of Agarose? As written before, there are many different types with different characteristics (EEO values, strength, melting point, etc.)for different purposes.
I'm not sure if the current bottle is from the same batch, probably not since I'm in a different country now. Storage condition may differ. It's way more humid here, though air conditioned. Also, the current bottle was in the fridge when I found it whereas we used to store it at room temp at my last lab.
The gel chamber is a bit different by I think both are by Bio-rad. Size wise, they are similar if not identical. The agarose are the same type from the same company (Sigma, Agarose for routine use). Maybe I'll get some from the next lab and see if there's a difference. I think I'll get someone from my last lab to check the dimensions of the chamber for me too.
Is this a different brand of ladder than you used in your old lab? And have you tried a different brand of ladder in your gels? I recently bought a new, cheaper ladder and that's exactly what happened to me - and their technical support wouldn't respond to my emails!