Hi, I'm trying to label an RNA ladder (RiboRuler high range from thermofisher) but I get this smear (see image), which I cannot get rid of, perhaps someone has any suggestions as to what causes the problem. I use the following protocol:
1.Treat the ladder with phosphatase (FastAP) to remove the 5' phosphate (using manufacturer's instructions).
2. Purify dephosphorylated RNA with Zymogen RNA Clean & Concentrator-25 colum.
3. Take the purified RNA and label it with PNK (according to manufacturer's instructions, using in buffer A).
4. Purify labeled RNA ladder with Sephadex G-25 column (also tried with Zymogen RNA Clean & Concentrator-25)
5. Resuspend ladder in 90% formamide containing loading buffer heat to 80C and cool on ice.
6. Run the sample in 3% 7M PAGE-Urea gel.
The image shows the resulting phosphoimage of the gel. Both lanes are the labeled ladder but loaded at two different concentrations. Based on the Silver staining I also know that the point where the signal starts to be very strong is the point where the top band of the ladder should run.
My guess is that the RNA has become degraded. Have you taken precautions to eliminate all RNase activity?
It is difficult to eliminate all free 32P but I think your problem is the gel. I did many agarose or polyacrylamide gels, but never 3% Page. I use to do agarose gel of concentration max 3-4% (difficult to melt) and UREA-PAGE minimum 6% 19:1 acrylamide-bis used for sequencing gel. In agarose gel you do not need to add formaildeyde if you denature the samples with 20 mM MOPS, 1mM EDTA, 5 mM NaAcetate, 50% (v/v formamide, 2.2 M formaldehyde), 10%glycerol, 10 min at 50° . I used to add in loading buffer 1ul of EtBr 1mg/ml
I agree with both Adam and Paola. Besides that, it could be due to carryover of reagent that presents in the gel.
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