1 reply to this topic

[lab_panda]

Hi,

I tried running glyoxylated RNA (1 ug and 10 ug) as well as DNA ladder (1 ug) on a SYBR safe gel in 1X MOPS buffer. I actually tried running on an agarose gel (1%) with SYBR safe and running a clean agarose gel first then staining with SYBR safe (2X in 1X TBE pH8.0). However, I get no bands for my glyoxylatd RNA and DNA ladder while I get bands for untreated RNA and DNA ladder of the same amount.

I tried asking Invitrogen about SYBR safe and glyoxal, they claim that SYBR safe behaves like EtBr and would react with glyoxal, which I think could be solved by staining the gel after running (which I tried but got no bands). I would like to ask if anyone have the experience with SYBR Safe and glyoxylated RNA. Would changing the buffer to sodium phosphate buffer pH 7 (instead of 1X MOPS) be helpful? Is it that glyoxal is actually interfering the interaction of SYBR Safe with DNA? I am using this glyoxylation method because I wanted to use a DNA ladder as a reference for my RNA size (as I do not have RNA ladder in my lab). (BTW the purpose of running the gel is just to check the approximate size of RNA and NOT for Northern blotting)

Thanks

[seanspotatobusiness]

Hey buddy. According to our Molecular Cloning, third edition, volume 1, page 7.27, "Staining glyoxalate RNA in agarose gels has also been a problem until recently. Staining the gel with ethidium bromide after electrophoresis is insensitive because of high background...". They go on to say that RNA can be stained with EtBr during denaturation with glyoxal. They say this will reduce hybridisation efficiency, but since you're not blotting, this is surely not a problem.

I think SYBR Maybe-Slightly-Safer-But-Probably-Not is more sensitive to heat, than EtBr, so I'm not sure whether you can include it in the denaturation step. I'd suggest just using EtBr. The only time I use SYBR MSSBPN is when I'm cloning and don't want to expose my DNA to UV.

Do you have access to the Molecular Cloning volumes? We keep ours in the lab, on special pedestals, and only the ordained can touch them.

Edited by seanspotatobusiness, 01 February 2011 - 03:46 AM.