Nupage Bis/Tris Current Voltage problem - Current is so high! (Mar/22/2010 )
Sharky on Mar 30 2010, 11:29 AM said:
fishdoc on Mar 30 2010, 07:20 AM said:
Sharky on Mar 30 2010, 08:51 AM said:
Denny on Mar 25 2010, 04:08 PM said:
Sounds like a good plan 
The Hall of Shame site is a riot!
The Hall of Shame site is a riot!
I dont believe it Denny! I used deonized, distilled water, made sure I did not overload, ascertained that Bugbuster reagent in the sample is not high salt content and is suitable for electrophoresis, and I am stil getting this high current problem and consequent frowing effect.
I noticed that unless the outer and inner chamber MES buffer touch, the voltage is 200, but the current is like 8-10 and very few bubbles can be seen. If its very low to 0 ma it means the process aint working right?
Do you use the Xcell SureLock cells for electrophoresis? I am wondering, because if you do I'm not sure what you mean by the outer and inner chambers touching. How much buffer are you putting into the inner and outer chambers?
Hey Fish, I dont use the Xcell SureLock. I use the Cleaver Scientific CVS10D Electrophoresis apparatus. I put enough inner buffer to cover the wells, and the remainder in the outer. Its about 200ml / 800 ml respectively. The weird thing is, unless I put enough on the outside to make the buffer connect, I get pretty much no current(around 16ma)
I run about the same volumes of buffer using the SureLock apparatus. The inner chamber is always full to the point that some buffer runs out. The outer chamber has been filled all the way up to just past the bottom horizontal line on the well marks, but the inner and outer never touch each other. I've also run gels with outer buffer only about 1/4 to 1/3 full, and didn't have any problem. Like I've said, I don't know what my mA was (is) for those runs, but haven't had any problems at all with the Nupage gels. The only problem I remember with the gels is when a student put the gels in the rig backwards, such that the two short plates (where you load your sample into the well) faced outward instead of facing inward toward each other. I remember there were very few bubbles, but I don't remember what happened with the samples, other than they didn't run right.
-fishdoc-
sharky,
I'm unfamiliar with your gel rig, fishdoc has much more info in that area for you. If you're not seeing a nice flow of bubbles, something's amiss with your set-up. I don't know. Sorry.
fishdoc on Mar 30 2010, 01:00 PM said:
Sharky on Mar 30 2010, 11:29 AM said:
fishdoc on Mar 30 2010, 07:20 AM said:
Sharky on Mar 30 2010, 08:51 AM said:
Denny on Mar 25 2010, 04:08 PM said:
Sounds like a good plan
The Hall of Shame site is a riot!
The Hall of Shame site is a riot!
I dont believe it Denny! I used deonized, distilled water, made sure I did not overload, ascertained that Bugbuster reagent in the sample is not high salt content and is suitable for electrophoresis, and I am stil getting this high current problem and consequent frowing effect.
I noticed that unless the outer and inner chamber MES buffer touch, the voltage is 200, but the current is like 8-10 and very few bubbles can be seen. If its very low to 0 ma it means the process aint working right?
Do you use the Xcell SureLock cells for electrophoresis? I am wondering, because if you do I'm not sure what you mean by the outer and inner chambers touching. How much buffer are you putting into the inner and outer chambers?
Hey Fish, I dont use the Xcell SureLock. I use the Cleaver Scientific CVS10D Electrophoresis apparatus. I put enough inner buffer to cover the wells, and the remainder in the outer. Its about 200ml / 800 ml respectively. The weird thing is, unless I put enough on the outside to make the buffer connect, I get pretty much no current(around 16ma)
I run about the same volumes of buffer using the SureLock apparatus. The inner chamber is always full to the point that some buffer runs out. The outer chamber has been filled all the way up to just past the bottom horizontal line on the well marks, but the inner and outer never touch each other. I've also run gels with outer buffer only about 1/4 to 1/3 full, and didn't have any problem. Like I've said, I don't know what my mA was (is) for those runs, but haven't had any problems at all with the Nupage gels. The only problem I remember with the gels is when a student put the gels in the rig backwards, such that the two short plates (where you load your sample into the well) faced outward instead of facing inward toward each other. I remember there were very few bubbles, but I don't remember what happened with the samples, other than they didn't run right.
-Denny-
I'm running a NuPage 4-12% bis tris gel right now in mops SDS buffer, and the mA is about 180-190 with a voltage of 190.
-fishdoc-