Hang Up in the Wells — Preventing Samples from Remaining in the Wells of Your Gels
|Scientists occasionally report that some of their nucleic acid samples remain in the wells of denaturing agarose and acrylamide gels during gel analysis. The problem is usually sporadic and not associated with a specific technique (probe purification, Northern blotting, nuclease protection assay analysis, etc). Often this problem can be overcome by measures as simple as switching brands of sample tubes, or taking more time to insure that samples are thoroughly resuspended. However, there are certain circumstances that do appear to reproducibly lead to sample hang up. While there is no ultimate solution, this article points out some of the special situations when "sample hang up" occurs, and provides suggestions for preventing "hang up" of your samples.|
Washing and Resuspending Pellets
Flushing the Wells of the Gel
Infrequently, despite care in pellet resuspension and flushing of gel wells, labeled material will still become stuck in the wells. We have found that an occasional Eppendorf tube will contain residue that can cause this to happen. Figure 1 shows a radiolabeled RNA probe that was aliquotted to 11 fresh Eppendorf tubes containing gel loading buffer, heated, and subsequently loaded onto a thoroughly flushed denaturing polyacrylamide gel. Two of the samples remained in the wells. Note that another sample was totally degraded, suggesting that an occasional tube may contain some ribonuclease. If a percentage of your samples continually remains in the wells of the gel, or shows degradation irrespective of the technique being used, we suggest that you try changing the supplier of your sample tubes. Autoclaving silanized Eppendorf tubes also appears to leave residue that can cause well hang up.
Gel purification of probes used in nuclease protection studies can help eliminate radioactivity left in the wells. Much of this signal is presumed to be due to residual unincorporated radiolabeled nucleotides that stick to the wells, as the intensity of this signal is usually independent of that produced by the target hybridized to probe. Figure 2 compares a mouse ß-actin RNA probe precipitated twice with NH4OAc and EtOH after synthesis to a probe purified on a gel. Both probes were then used in a ribonuclease protection assay to protect ß-actin transcripts in 10 µg of total mouse liver RNA. When the digestion products were loaded on a gel, the sample wells in which non-gel purified probe products were loaded showed residual radioactivity. Note, however, that there is no loss of protected fragment signal in these lanes compared to those in which gel purified probes were used.
Carriers and Protein Molecules
Protein and carrier molecules can lead to hang up in the wells. Crude RNA preparations containing significant amounts of protein or chromosomal DNA can form insoluble complexes upon precipitation that remain in the wells. Proteinase K treatment followed by phenol extraction or DNase treatment will often dissolve these complexes. However, addition of enzyme to the sample to degrade contaminating protein or DNA can also produce this effect. Figure 3A shows a gel where proteinase K has been added to replicate aliquots of an RNA probe transcript. As the concentration of proteinase K is increased (from 0 µg proteinase K in lanes 1 and 2, 35 µg in lane 3, to 70 µg in lane 4), the amount of radiolabel remaining in the wells also increases. Note again that the probe transcript signal remains unchanged.
Linear acrylamide, glycogen, RNA and DNA are all used to enhance quantitative precipitation of small amounts of nucleic acids from dilute solutions. Since linear acrylamide is the only one of these molecules purified from a non-biological source, it is considered the carrier of choice when used in downstream applications in which contaminating nucleic acids could yield spurious products (PCR and RT-PCR applications) or compete for labeling (end-labeling with terminal transferase). However, linear acrylamide can cause well hang up, whereas RNA and DNA carriers do not (Figure 3B).
10% ammonium persulfate (APS) aliquots can be purchased in gelatin capsules as a polymerization catalyst for polyacrylamide gels. However, in Figure 4, Dr. Scott Nelson (University of Iowa, personal communication) demonstrates that these capsules seriously impeded sample migration. When increasing amounts of Ambion's RNA Century Markers were run on a denaturing acrylamide gel made with the 10% APS capsules (Panel A), their migration pattern was strongly affected. However, when the same dilution series was run on a gel made with fresh 10% APS, the size markers migrated as expected (Panel B).