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Frequently Asked Questions: RiboRuler™ RNA Ladders (5)
- RiboRuler™ RNA ladders and other RNA related products
- 2X RNA Loading Dye
- RNA quantification on gels
- RNA electrophoresis
- Northern blots and labeling:
- Can RiboRuler™ RNA ladders be used for Northern blots?
- Can Fermentas SensiBlot™ Plus Nylon Membrane be used for Northern blots?
- What quantity of the ladder should be loaded to successfully transfer it onto the membrane?
- What electrophoresis conditions are the best for Northern blots?
- Can RiboRuler™ RNA ladders specifically or non-specifically hybridize with my probe?
- Does the ethidium bromide included in the 2X RNA Loading Dye interfere with RNA transfer or hybridization results?
- Can RiboRuler™ RNA ladders be radioactively labeled?
- Is it necessary to dephosphorylate RiboRuler™ RNA ladders prior to labeling of 5'-ends?
1. Can RiboRuler™ RNA ladders be used for Northern Blots?Fermentas RiboRuler™ RNA ladders are ideal for Northern Blots and result in sharp discrete bands on membranes.
RiboRuler™ RNA Ladders (#SM1831 and #SM1821) can be labeled radioactively with T4 Polynucleotide Kinase.
2. Can Fermentas SensiBlot™ Plus Nylon Membrane be used for Northern blots?SensiBlot™ Plus Nylon Membranes are ideal for Northern blots.
3. What quantity of the ladder should be loaded to successfully transfer it onto the membrane?A 2 µl aliquot (0.25μl of the ladder for every mm of the gel lane width) of the RiboRuler™ Low Range RNA Ladder and RiboRuler™ High Range RNA Ladder
or a 4 µl aliquot (0.5μl of the ladder for every mm of the gel lane width) of the RiboRuler™ Low Range RNA Ladder, ready-to-use and RiboRuler™ High Range RNA Ladder, ready-to-use is easily visible after transfer onto a SensiBlot™ Plus Nylon Membrane from a 1% formaldehyde gel.
4. What electrophoresis conditions are the best for Northern blots?For the most complete RNA denaturation, perform electrophoresis in a denaturing formaldehyde agarose gel in MOPS buffer.
Native agarose gels require higher quantities of RNA to ensure that adequate transfer occurs.5. Can the ladders specifically or non-specifically hybridize with my probe?RiboRuler™ RNA ladders are mixtures of chromatography purified single-stranded RNA transcripts, produced from specific templates that contain a fragment of the pTZ19R polylinker and Lambda phage fragments. Certain probes may hybridize with RiboRuler™ RNA ladders.
Since each ladder band contains a high concentration of RNA, non-specific hybridization with other probes is also possible. Because of this, the ladder is sometimes visible after hybridization.6. Does the ethidium bromide included in the 2X RNA Loading Dye interfere with RNA transfer or hybridization results?Ethidium bromide as present in samples prepared with 2X RNA Loading Dye (at a final conc. of 0.0125%) does not interfere with either RNA transfer onto the membrane or with RNA hybridization to probes. There are some reports in the literature that ethidium bromide, when used at high concentrations, may interfere with RNA transfer efficiency or hybridization. However, this issue is isolated to cases where ethidium bromide is used both in the gel and in the electrophoresis buffer during the gel run.
Loading dyes containing ethidium bromide should not be used in cases where very small populations of the target RNA are present.
7. Can RiboRuler™ RNA ladders be radioactively labeled?Yes, for radioactive labeling with T4 Polynucleotide Kinase, conventional versions of ladders: RiboRuler™ Low Range RNA Ladder and RiboRuler™ High Range RNA Ladder are ideal.
Ready-to-use versions (RiboRuler™ Low Range RNA Ladder, ready-to-use and RiboRuler™ High Range RNA Ladder, ready-to-use) are premixed with the electrophoresis loading dye and are not suitable for enzymatic manipulations.8. Is it necessary to dephosphorylate RiboRuler™ RNA ladders prior to labeling of 5'-ends?RiboRuler™ RNA ladders are composed of 5'-phosphorylated transcripts. Since the forward 5'-end labeling reaction with T4 Polynucleotide Kinase is always more efficient than the exchange reaction, it is recommended to dephosphorylate the ladder or any other RNA transcript prior to labeling.
An additional benefit is that the forward labeling reaction requires only half of radioactive label needed for the exchange reaction.
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Updated vasario 01, 2008 10:41
