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polyacrylamide gel electophoresis - (Feb/24/2007 )

Hi,

I am trying to resolve RNA oligonucleotides of about 20bp. I purchased precast gels from Biorad. They are 15% TBE gels. When I load the wells and electrophorese, the products remain in the gel. Does anyone know why? Is it the sample, the loading buffer, or the gel itself? I used 1X TBE running buffer, 100V for 60mins to run the gel.

Thanks,
Sybr

-sybr-

QUOTE (sybr @ Feb 24 2007, 02:23 PM)
Hi,

I am trying to resolve RNA oligonucleotides of about 20bp. I purchased precast gels from Biorad. They are 15% TBE gels. When I load the wells and electrophorese, the products remain in the gel. Does anyone know why? Is it the sample, the loading buffer, or the gel itself? I used 1X TBE running buffer, 100V for 60mins to run the gel.

Thanks,
Sybr

do you mean "remain in the well"? they should remain in the gel (and get stained to visualize).

-mdfenko-

Sorry,

The samples remain in the well. They do not migrate. When stained with EtBr, they are in the wells still, even after running for 1 hour.
Thanks,

Sybr

-sybr-

Do the dyes remain in the wells also?

-tfitzwater-

Yes. dye remains in the well. The wells appeared bright after staining, so product (RNA) is there too.

Still I have no solution as to why they did not migrate.

Sybr

-sybr-

The most likely explanation is that you are using water rather than 1x TBE for running buffer. Prepare a fresh batch of 10x TBE or get some from another lab to check for this problem. I have observed a case in another lab where one person was tired of constantly having solutions "borrowed," and replaced his TBE with water in order to get back at the others in his lab. There is a chance that Bio-Rad left out the TBE when they formulated the gels, but this is doubtful. If the buffer is not the explanation, then try another power supply.

Preload your gel with loading dye only to make sure everything is working and the dyes enter the gel before you load your RNA samples.

-tfitzwater-

I'm with tfitzwater on this. Had the same once with TAE and agarose gels, made fresh 50x, diluted to the right concentration and problem was solved.

-vairus-

Thanks. I will try a new batch of 1 X TBE buffer.......even though I did see bubbling in the tank, indicating that the power was applied. Only thing done differently was that the 50X buffer was diluted to 1X in RNase free distilled water.

Thanks
SYBR

-sybr-

TAE buffer is typically 50X while TBE buffer is normally 10x. If you diluted 10x TBE 50-fold down to 0.2x it might still work, since 0.5x TBE works OK, but I have never tried.

-tfitzwater-