2 replies to this topic

[wjchxl]

Hi,

I am going to use a 10bp DNA ladder from Promega as a size marker, which consists of 10 blunt-ended DNA fragments ranging from 10bp to 100bp in exactly 10bp increments.  However, my samples will be radioactively labeled, and the gel is going to be analyzed with PhosphorImager.  Can the DNA ladder be radioactively labeled at the end?  If yes, could anyone suggest a protocol?  Thanks a lot!

[tanyaaspinall]

I don't know a way to radiolabel the marker. When I do protein gels, I use a prestained marker. You can see on the gel where the markers are without staining. I usually dry my gel down, then spot a bit of my radioactive sample onto 2-3 of the markers. After drying these onto the gel (with a hairdryer!), you can phosphoimage and will see the labelled markers.

For a DNA gel, you could ethidium bromide stain and dry your gel, then spot on a little of your radioactive sample onto a couple of the marker bands.

Hope this helps!

[badcell]

Hi. I guess you could label the markers with T4 polynucleotide kinase, as they probably will be not phosphorylated at the ends. I've used this method sometimes to label single- or double-stranded oligos for EMSA of footprinting, but I don't have a written protocol. Here's the link to the NEB T4 PNK1, which is the enzyme I have used. It has a short protocol on it.
Hope this helps!