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Problems with having to run the gels for so long


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#1 alejandro LSU

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Posted 23 September 2012 - 09:06 AM

Im having an issue with my electrophoresis. For some reason the DNA is not moving down the gels as fast as it should. Im having to run the gels for about 20 hours (instead of the normal 4-5 hours). Does anyone know what the reason is or could be?

#2 Trof

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Posted 23 September 2012 - 12:07 PM

What gel percentage and what DNA size? 4-5 hours is not normal, unless you have extra long fragments on a low voltage or something.
Your power source can be bad, your electrodes/tank can be bad. Try to change both. Does your gel heat during run even on low voltage? That could mean increased resistance through bad wiring/electrodes, gel or buffer. If the gel is running slow and is cool, and also bands are diffused, you may just not get enough current because of malfunctioning power source.
If you change both tank and power source and it's still slow, try to find problems in buffer and gel composition.
No more ideas for now.

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#3 bob1

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Posted 23 September 2012 - 12:21 PM

I would also suggest making fresh buffer - try switching to SB (sodium borate) or LB (lithium borate) buffers, they run much neater and faster for many sizes of DNA.

#4 alejandro LSU

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Posted 23 September 2012 - 12:52 PM

I use 4.5% gel, I think, and run at 100 volts. Im doing a gene mapping study so i need to run the gels enough so that I can tell when the polymorphic markers separate

#5 alejandro LSU

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Posted 23 September 2012 - 12:53 PM

It used to get warm when it worked fine but now its always cool.

#6 ascacioc

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Posted 24 September 2012 - 12:32 PM

make new buffer and check whether the electrodes are fine. For that I would check the current (mA) to check whether the tank is not short circuiting.

Andreea

#7 alejandro LSU

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Posted 24 September 2012 - 12:51 PM

how do I check the electrodes, and what should the mA be around?

#8 ascacioc

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Posted 24 September 2012 - 12:54 PM

Check electrodes = you check whether you have bubbles in the buffer when you start the run; check whether they are not loose
mA should not be 1 mA. I am not familiar with your gel system to tell you exactly. But you shouldn't get too low current. Check and tell me how much you get. For your gel composition I would expect smth around 10 mA (but I might be wrong)

#9 bob1

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Posted 24 September 2012 - 12:55 PM

how do I check the electrodes, and what should the mA be around?

Find a multimeter or something that can measure resistance - touch the probes to each end of the electrodes and/or leads and read the reisitance - if it approaches 0, then they are fine.

Short circuiting will lead to very high current.

#10 ascacioc

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Posted 24 September 2012 - 12:59 PM

Actually bob is right: short circuiting leads to high current. Anyhow.

Your current should not be 1 mA and not too much. Now, it would have been nice for you to know what the current should be from a nice run. Next time when you have a nice run, check the current and note it down. It might be handy to know it for next time when you have troubles. :)

#11 alejandro LSU

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Posted 24 September 2012 - 01:08 PM

I usually run two gels on one Power Pac. That hasn't been a problem before but I don't know if its a problem now.

#12 ascacioc

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Posted 24 September 2012 - 01:14 PM

2 gels means that you should have double the current. Try to see each gel alone what current you get.
Do you have the same phenomenon in both gel aka not running? Then it is the power pack or smth else that is in common for both gels. Did you redo the buffer?

#13 alejandro LSU

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Posted 24 September 2012 - 01:20 PM

Yeah the problem is the same for both the gels in the two separate tanks. I making new buffer to try it out tomorrow. Thanks for all the help.

#14 ascacioc

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Posted 24 September 2012 - 01:26 PM

you might also want to try a new power pac if the new buffer does not work because I do not think that the electrodes in both tanks got ruined in the same time. Good luck for tomorrow :)

#15 phage434

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Posted 27 September 2012 - 02:26 PM

Some power supplies will shut down if the current is too high. Running with both gels might cause this.




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