I have DNA gel casters of two dimensions, 7X10 cm and 15X10 cm. The small gel is working fine but I am facing problem with the big gel. By some mysterious reason I never get DNA band, even ladder on big gel. My agarose, 1x TBE buffer, EtBr, ladder, running condition all are same for both small and big gels. I don't know which condition I have to alter for big gels.
Just a quick question, do you leave the gel in the gel tray when you are visualising the gel on the UV bench?
I know that some gel trays absorbe UV light and therefore, the bands are not visible.
If you are leaving the gels in their trays that could be an explanation.
This is almost one of those little things that you're going to kick yourself for once you figure it out!
Are you scaling the volume of EtBr with the volume of agarose/TBE? How far does your DNA loading buffer go into the gel? Is it maybe running right through? Have a look at it 5 minutes after you start the power and see that it's going in the right direction! (I had a colleague that once "inadvertently" switched the pos/neg leads for one tank).
As William says, some casting trays don't allow UV through. Take the gel off the tray to visualise.
Dear All,
Thanks for the points you have highlighted. Here I am adding few of the facts related to this big gel...
1) For this big caster (15 X 10 cm) , I generally prepare 60 ml of 1% agarose solution, with 4-5 microlit. EtBr (10mg/ml stock).
2) Wells are wide (0.7 mm).
3) For DNA checking and PCR products I usually load 4-5 microlit of samples with 2 microlit of orange dye (6x conc.). My ladders are diluted, so I load 7-8 microlit/well.
4) The facts stated in third point are working well for small casters (7 X 10cm), whereas for big caster I am consistently getting the visibility problem.
5) I have tried with different agarose, I have changed buffer but no use. I have one doubt whether EtBr longivity matters. My EtBr solution is old.....Is it the culprit????
- Badges
- Science method