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Smeary bands on agarose gel (also the ladder) - (Dec/02/2013 )

Hi,

 

Firstly..I'm new on this forum and I'm sorry if this question/problem is solved for several times on previous threads smile.png

 

I'm quite experienced with lab work but this is the first time when I counter this problem:

 

I've been running my digested vector on 1,2% gel with 1 x TBE. I intend to purify the linearized vector from the gel with commercial kit. Anyway..  for some reason all bands on the gel (including the ladder) appears to be very smeary. This has happened now for couple of times in a row. I run the gel 5V/cm (which should be just fine). Today when I was laying the gel on the UV-table I noticed that the gel was strangely warm. All my reagents should be fine, but I have some doubts with my TBE. Could "bad" running buffer somehow increase the current of the system resulting to the heated gel and degreaded (smeary?) samples ? My dye is Gelred.

 

Thanks!

 

 

-tmaz-

There could be a number of things wrong, the usual causes of smeary bands are: degradation (won't be this unless you contaminated your ladder also), too much DNA (how much did you load?), buffering capacity of the electrophoresis buffer (check pH and salt content), and too much salt in the DNA (what was the source and how prepared?)

-bob1-

There could be a number of things wrong, the usual causes of smeary bands are: degradation (won't be this unless you contaminated your ladder also), too much DNA (how much did you load?), buffering capacity of the electrophoresis buffer (check pH and salt content), and too much salt in the DNA (what was the source and how prepared?)

 

Thank you for the reply!

 

The problem dissappeared when I stained the gel after running it. Previously I added Gel red to the molten agarose/TBE. Still... a bit weird I think.

-tmaz-