big problem with transfer - reversed polarity? - (May/03/2008 )
I've always done Westerns using the mini cells from biorad for running the gel and tranfering, but this week I tried to use the larger transfer blot cell from Biorad. I know I made the "sandwich" right and connected the power cords properly (I was beign very careful to do so!), and ran it at 60V overnight, with the cooling thing in. When I came in the next day, the buffer and cathode were orangey brown (presumablly rust), the filter pads, gel and membrane were rusty too, and the ladder was nowhere to be seen. Ponceau of the membrane showed nothing and, again there was no ladder left in the gel. I thought I must have made my buffer wrong, adn washed out the apparatus although some rust wouldn't come off. I tried this again using newly made buffer, and the same thing happened! The manual at one point warns not to reverse the polarity of the electrodes or the cathode might rust. Does anyone have any idea of what could have happened and how to solve it?
Hi, presumibly you roasted your system! i use 90mA (i keep Amperage as constant) for overnight WB transfer, and 250-300mA for a 90min transfer. When i use 250mA for 90min, the Voltage doesn't exceed the 20Volts.
So, i suggest you to keep your transfer at 90mA in constants amperage overnight, without cooling system (don't care for overnight transfer). I normally use tris/glycine buffer with 10% Methanol, 0.03% SDS.
i hope this may help you,
let me know if it works