Metaphor agarose optimization - (Sep/01/2006 )
I am currently trying to resolve DNA fragments (around 100 to 300bp large) which differ by 2 to 6bp. I am using 3% Metaphor agarose 0.5X TBE to resolve my fragments and used to get really nice and sharp bands ( check out the first photo) but recently, I have been getting crappy, smeary bands (check out the second photo). I thought that it could be that my PCR products weren't clean or smtg but the thing is, my ladders aren't that clear either...
I have tried
1. using a lower concentration of TBE (0.5X instead of 1x to reduce overheating)
2. running it at low voltages (85V 3h instead of 100V)
3. leaving my gels to set for more than 1 hour before refrigeration to ensure proper setting
4. ensuring that buffer in my gel tank is of the same batch and concentration as the buffer used to make my gel
but so far, I have been unable to replicate what I used to get. I now get slightly smeary bands which are a pain cos I am doing fingerprinting with microsatellites so a slight difference in band size would equate a different allele... smeary bands just give me a headache
so guys, any suggestions/ past experiences?
p/s my gel tank does not allow me to keep my buffer temperature at a constant temp.. so high voltages tend to cause my gels to overheat...
Some things to check include the loading buffer and the F-stop on the camera. The depth of field of the camera is controlled by the F-stop, and you should set it as high as possible, which will keep more of the field in sharp focus.