I am new to sds gels and western blotting. My samples, ladder and just blank loading buffer will not run at all, they just sit in the well and do not move in the slightest. I got my gel and reagent protocols from another lab who runs these constantly. I tried using their loading buffer so I have ruled that out as the cause (plus the ladder is just directly added to the well). They double checked all my calculations for the reagents and cannot tell me what might be wrong. I see bubbles when I turn on the voltage and all of my gel prepping reagents are brand new or freshly made. Is this issue definitely in my gel preparation, or could it be the running buffer or gel apparatus?
It sounds like there's no current running through the gel Check if the power supply has the right voltage (100-300 V) and is pushing current. Did you remove the tape or other packing material from the bottom of the gel? Does the buffer cover both ends of the gel? Make sure there is no alternate path for the electricity to flow around the gel.
I tried two different power suppliers and two different gel apparatuses. I made the gel, so there would be no packaging. The buffer is covering the top. How do I check to make sure there is no alternate path for the electricity?
Thank you.
Make sure your +ve and -ve wires go into the right ports, or current will flow in the opposite direction and your protein wont move.
hello Butler
if you could share the protocol for your gel preparation and loading buffer preparation it will be helpful for trouble shooting.
Regarding alternate electrical paths, make sure there's no connection between the upper and lower buffer tanks.
I'm at this stage too! Except I find no bubbles when I turn on the power! I'm using 1x TBE for DNA seperation, but have no idea whats wrong. One thing I am trying right now is to heat up the buffer to around 40 degrees celcius and then load it into the top and bottom reservoirs
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