SDS-PAGE run problems
Posted 18 January 2005 - 07:56 AM
Although everything looks fine (voltage 200V etc) the samples donīt seem to get out of the wells. Indeed after some time the dye begins to blur and disapear. After almost an hour of run I donīt even see the front line.
Thanks a lot.
Posted 18 January 2005 - 08:10 AM
Posted 18 January 2005 - 08:19 AM
Brand new apparatuses often have conductivity issues. Make sure there is current flowing through the system. A Tris-glycine buffer filled system should show at least 25 mA at 100 V- if not, something is wrong.
Posted 18 January 2005 - 08:55 AM
It has 125 mA.
Is it possible that buffer pH could induce this? I've just measure them again and they are a bite lower than they should...
Thanks a lot.
Posted 19 January 2005 - 06:36 AM
The current is too high. This suggests that either gel conductivity is high because of ions in acrylamide (improbable - but happens once in a while) or there is another electric contact being made between buffer reservoirs separate from the gel sandwich (highly possible). Your samples donot move because all the current is not passing through the gel - it is being diverted somehow. Try this- remove the gel sandwich- connect apparatus to power. If the current is still at 125 mA at the set voltage - then the gel is not seeing the current at all.
Posted 19 January 2005 - 06:39 AM
If the buffer is bad- then the gel will either run slow or very fast - but not diffuse like you describe. If I were a gambling person, my money would be on the apparatus, not on the buffer or your samples.
Posted 20 January 2005 - 07:39 AM
I did what you mentioned on your last post and as you predicted it do seems that the current is not passing thru the gel!
I'll just have to check the apparatus for the problem...
Thanks a lot, you were very helpfull!
Posted 20 January 2005 - 09:18 AM
You are very welcome. All we have is each other in this often times very difficult profession.