DNA agarose gels - trouble shoot weird gels (May/23/2006 )
hi
have some trouble with agarose gels.Pls have alook at the attached picture.
i use a 0.8% agarose gels.run with 1x TAE buffer.
1. Why the loading dye forms patches ,dark patches.use a 6X loading dye with xylene cynol&bromophenola blue.Use as 1/6 of the total loaded volume.Anything wrong with it.Its a quite old loading dye stored at -20 for a couple of yrs.
Should i leave the loading dye to come to room temperature or should use immediately.Does this have any impact on the dark regions?
2.some of the bands are weird., esp the last lane it forms a thin line from the wells, why is this happening.
pls pass ur suggestions.Will be useful to trouble shoot.
have some trouble with agarose gels.Pls have alook at the attached picture.
i use a 0.8% agarose gels.run with 1x TAE buffer.
1. Why the loading dye forms patches ,dark patches.use a 6X loading dye with xylene cynol&bromophenola blue.Use as 1/6 of the total loaded volume.Anything wrong with it.Its a quite old loading dye stored at -20 for a couple of yrs.
Should i leave the loading dye to come to room temperature or should use immediately.Does this have any impact on the dark regions?
2.some of the bands are weird., esp the last lane it forms a thin line from the wells, why is this happening.
pls pass ur suggestions.Will be useful to trouble shoot.
I don't think there is anything wrong with the dark patches. I get the same thing, It has something to do UV light and dye interacting. The last lane may look weird because it's at the edge of the gel. If I can help it I usually don't load anything into the 1st and last lanes.
I think the dark patches are actually places where there is no ethidium bromide left in the gel. (if you used EtBr) Try adding some to the tank and to the gel.
Also, somethimes the lanes get funny looking if the gel is run too fast. try slowing it down. ( use a lower voltage for a longer time)
To me I would say:
- a little bit too much dye in your loading mix. If it is home made, change recipe with less dye.
-Your picture look as if you do not put the orange filter between gel and camera, that would greatly reduce background, and I bet improve your picture.
maybe try other dye, orange G ... runs very fast 
@mini
I get dark regions too if i use handmade dye. But, it lighter if i use not handmade dye
band like 'U' it happen, maybe caused by too much sample that you load on lane.
try to submerge the gel for 20 minutes in distilled water for better picture.
try it for good background on your gel
have some trouble with agarose gels.Pls have alook at the attached picture.
i use a 0.8% agarose gels.run with 1x TAE buffer.
1. Why the loading dye forms patches ,dark patches.use a 6X loading dye with xylene cynol&bromophenola blue.Use as 1/6 of the total loaded volume.Anything wrong with it.Its a quite old loading dye stored at -20 for a couple of yrs.
Should i leave the loading dye to come to room temperature or should use immediately.Does this have any impact on the dark regions?
2.some of the bands are weird., esp the last lane it forms a thin line from the wells, why is this happening.
pls pass ur suggestions.Will be useful to trouble shoot.
HiI had the same problem
Try using orange day ,it doesnt leave any dark spots!!!!!!
I you re-stain your gels with SYBR gold it will get rid of the blue bands. Found this out the other day.