I am running an agarose gel to check for RNA degradation, and I'm having trouble getting my sample to migrate out of the well. I've attached a copy of the picture showing the sample in the well.
I'm using 1.5 uL RNA + .5 uL RNase free H2O and 2 uL of 2X gel loading dye.
I appreciate any suggestions or help,
Kimberly Baker
By the way it seems like the electrophoresis machine and apparatus has some problem. Check it in someone elses lab first. If the problems still the same, then check the following things:
>RNA concentration (by nanodrop. Should not be more than 1000)
>electrophoresis Voltage and Current
>use 6-10x loading dye
>use 1xTAE (freshly prepared with ddH20), clean the apparatus too
> use 0.8-1x agar gel
> use one size bigger comb
Hi
Did you try with other samples with the same electrophoresis system?? First try with previously done electrophoresis samples. If the problem is also persists please follow the Muhammad Owais Shahid suggestions. I also agree with him.
Kim,
I agree with the above troubleshooting strategies as well. But in addition, if your RNA sample is very concentrated (which it appears to be), you may want to run several lanes with a dilution series of the same sample to see if there are any differences in their migration. If they all pool in the wells, then I would check your protocol. You may want to be sure you're denaturing your samples properly before loading them.
Thank you for the help, everyone! We got it to work by increasing the voltage to 90V and running it for longer.
I'll keep all of your input in mind for the future.
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