Posted 07 February 2010 - 02:10 PM
I've been facing a problem in DNA agarose gel electrophoresis. My low molecular weight bands ( and bromophenol blue dye ) appear as curve bands ( frowning not smiling ) . I am using a fresh 0.25x TBE . 2.5% agarose ( 20 cm x 20 cm x 5 mm ) . My agarose is prepared in also 0.25X . The voltage I am using is high ( 8 V/cm ) but the current is only 40mA so there's not any excessive heat generation . The electrophoresis run is short ( only 30 minutes ) . I tried different samples , fresh buffer , higher buffer concentration , lower voltage . But still the same . The bromophenol dye appears as a sharp curved band ( frown ) as soon as it enters the gel.
I get the same problem in all different lanes of the gel .
I wonder if it's because of poor gel preparation . I always get bubbles trapped in the gel ( very very tiny bubbles but there are thousands at some spots ! ) . However , the region ahead of the wells seems very clear so I don't think this is the problem .
I don't know what to do ...any help would be appreciated
thank you in advance
Posted 07 February 2010 - 03:43 PM
Posted 07 February 2010 - 03:55 PM
Thank you for your reply but the bromophenol blue band apears as a frown as soon as it enters the gel ( after 3 minutes ) . 3 minutes are not enough for heat to build up . Also , sometimes I get this issue in two or three lanes but not in all the lanes . The molecular weight marker weights always seem ok .
Usually a frown (or smile, depending on whether you are using agarose or PAGE) occurs because the gel heats up at the edges more than the middle (or vice versa) resulting in larger pore size in the matrix, allowing that edge (middle) to run faster. I would try using 1x TBE (there is a reason why you use it at 1x) or switch to SB buffer, which is more tolerant of higher voltages.
Could it be the ionic strength of the sample ?
Posted 08 February 2010 - 09:01 AM