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-   -   Sybr Green II and denaturing agarose gels (http://www.molecularstation.com/forum/rna-techniques-forum/3354-sybr-green-ii-denaturing-agarose-gels.html)

heliozoan 05-10-2008 08:42 AM

Sybr Green II and denaturing agarose gels
 
Hi,

I recently ran a Trizol RNA extract and Invitrogen's 0.5-10Kb RNA ladder on a denaturing formaldehyde agarose gel (1% agarose) in 1x MOPS buffer (following an Ambion gel recipe). Both extract and ladder were mixed with Sigma's RNA loading buffer at a buffer to sample ratio of 2:1. Sample and ladder were heated to 65C for 10 min., chilled on ice and then loaded on the gel. 3 micrograms of ladder was loaded. Upon completion of electrophoresis the gel was immersed in Sybr Green II prepared in 1x MOPS buffer at a dilution of 1:5000 per the Invitrogen/Molecular Probes protocol. After 45 min of staining the RNA bands were barely visible - even after a 30 sec. exposure with broad bandwith (290-365 nm) UV excitiation. I spoke with a tech at Invitrogen who suggested that the formaldehyde in the gel was quenching Sybr Green II - which is in direct contradicition of the product literature. He also suggested that the tracking dyes could interfere with the Sybr binding to the RNA. What are your experiences with using Sybr Green II to stain formaldehyde agarose gels? Recommendations?

Thanks!

danfive 05-10-2008 06:25 PM

Re: Sybr Green II and denaturing agarose gels
 
Formaldehyde shouldn't interfere, neither should bromophenol blue in the tracking dye. pH should be considered, SYBR range is 7.5 to 8.3 more or less and it loses sensitivity. Perhaps you should change to diluting in Tris buffer instead of MOPS.

Like this protocol (p.1) that has RNA run in MOPS buffer then stain in SYBR diluted in TBE ---> [Only registered and activated users can see links. Click Here To Register...]

I assume you've seen the useful tips from Molecular Probes for SYBR nucleic acid staining ---> [Only registered and activated users can see links. Click Here To Register...]

heliozoan 05-11-2008 05:23 AM

Re: Sybr Green II and denaturing agarose gels
 
Thanks for the links,much appreciated.

The pH of my 1X MOPS buffer used for both gel casting and as running buffer is supposed to be around 8.3 (Sigma MOPS-EDTA-Sodium acetate #M5755) so I thought that it would be OK for use with the Sybr Green II. It's interesting to note that in the linked protocol from the University of Alberta they do not use Sybr Green II for staining the formaldehyde gel, only in the formamide-only gel. The recipe for the formaldehyde gel at the U-Alberta link is the same one that I used for my gels. I found another formaldehyde gel recipe on the BioTechniques forum that only calls for 1.44 ml of 37% formaldehyde in a 100 ml gel, compared to 18 ml of 37% in the recipe I used.

I'll try staining in TBE anc cutting back on the formaldehyde.


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