When I'm running SDS-PAGE 12%, my sample moves to the other well (it's formed as a small curve with the following well) , even if I put it slowly,carefully, 15ul per well and I'm using a 1mm glass. I think it may be the sample buffer i use, it is dense. I look forward to your recommendations.
Sample buffer recipe (5x):
For 1ml:
- Tris (1M, pH 6.8) 0.25ml
- SDS 0.1 g
-Bromophenol blue 0.005 g
-Glycerol 99.5% 0.502 ml
- H2OMiliQ 0.25 ml
I use sample Buffer 1X
Are tou rinsing the wells with electrophoresis buffer just before loading the well mixed sample? During standing and pre run salts will come out of the gel and form a dense layer at the bottom of the wells
The density of the sample should be greater than the density of the solution in the well, so that the sample sinks when dispensed. Rincse out the wells with the upper chamber buffer before loading the samples, as suggested by Paul Rutland . If that isn't sufficient to solve the problem, make a denser sample buffer by increasing the concentration of glycerol.
I would really like to see a photo/image of what you are describing. It maybe a mix of both, sample preparation and the gel preparation. Just a few suggestions I can imagine to be the problem. Can your sample moving be described as floating out of the well? Can happen if there is DNA in your sample, which can be degraded by adding benzoenase or DNAse. Are the walls between the wells intact? Are there bubbles in your wells? Please rinse your wells as suggested by Adam and Paul. Do you use commercially prepared gels or make your own? If making your own, is there enough stacking gel between the bottom of the wells and the separation gel? Is there a good seal between the glass plates and the gel? Is there a possibility that your gel dried out some?
Good luck troubleshooting your gels!
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