2 replies to this topic

[RecklessTech]

Hey y'all

I'm running a reverse transcription-pcr rxn for amplicon (bp) sizes 213, 332, 407, 508, 586, 734, and 676. All are optimized for annealing and extension. The variation in size when running a 2% agarose gel is roughly 20 bps. Could this just be software variation? I feel like this is acceptable but my boss wants it tighter. Is there a reference for acceptable margins of error on gels? I appreciate your input in advance.

-RT

See attached

Attached Thumbnails

• 

Edited by RecklessTech, 29 November 2010 - 10:09 AM.

[hobglobin]

There's often a difference between lanes on sides and the middle of the gel ("gel smiling"), due to uneven temperature and electrical field within the chamber/gel. I guess it depends at least partly on chamber construction too, as it not occurs in different chambers to the same extent (high-tech gel chambers with temperature regulation anyway not).
Try to put the size marker in a middle lane (and perhaps right side) too, to have a more exact idea of the band sizes. If the software don't accept this (some even use several size marker lanes to interpolate between them), analyse it by hand.

Edited by hobglobin, 29 November 2010 - 10:48 AM.

[phage434]

If he really wants good resolution, then agarose is not the right answer.  You could always sequence them. But you can get higher resolution by running your gel longer (2.5x or so longer, I would guess).  You are destaining the gel with no EtBr in the running buffer, with the EtBr running toward the negative electrode.  Add stain to the positive electrode running buffer well.