Ive noticed that when im using the biorad mini system for western blot there tends to be a small amount of overflow into adjoining wells when im loading the samples even though im loading often times only 15ul in a 10well gel with 1mm spacer. I pipette the wells slowly and carefully but it looks like a small volume of the liquid rises up out of the well along the edges of the well and into the the next well. Does anyone know what causes this and is there a way to stop it?
It looks like your SDS-Sample Buffer does not have Glycerol which usually prevents the effects you described. You might want to include 10% Glycerol in your Sample Buffer.
you can load up to 20ul samples in 1mm spacer in 10 well . causing problem 1. The sample buffer or preparation gel and running buffer.
Beyond, do u use colibri tips? Also, some people use hamilton pippetes to load, and it is critical w/both to move upwards the tip so that the current goes down and does not bounce on the gel making currents upwards. One boss made me repeat my gels putting one empty lane between loaded lanes!!!
Use gel loading tips. As Pavel said while loading samples slowly keep moving the tip upwards to prevent the sample from spilling out of wells.
http://www.sigmaaldrich.com/catalog/product/sigma/cls4853
Hi Peter, I am assuming you are already using gel loading tips and the correct loading buffer, so there could be an issue with the pouring of your stacking gel. A couple of things can happen - if your clamps are not sufficiently tight (this can happen as they age), then when you pour your stack you get a thin layer of gel forming at the back of each of the teeth (I hope this makes sense) and this essentially makes the volume of your well smaller. the other issue can be that the gel separating the wells often "shrinks", so when you take the comb out they are not as tall as they should be - to avoid this, either pull the comb out a tiny bit after inserting it as this can help, or once inserted, top the well up along the comb. Finally remember to wash the wells very carefully and thoroughly before loading. I hope this helps. Let me know how you get on. Drop a line if you need anything else. Claire P.S. Not sure what concentration of Laemmeli buffer you are using, but if 2x, change to 5x and you will then be loading a smaller final volume.
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