The exquisite properties of Morpholinos contribute to their extremely high success rate, particularly as compared to Phosphorothioates or other unstable or nuclease-sensitive antisense structural types. However, occasionally Morpholino oligos can fail and there are many factors that can contribute to oligo failure, several of which are correctable once determined. Some of the factors that contribute to failure and solutions for many of these same problems are listed as responses to typical researcher concerns below.

1. I can't resuspend my oligos
• Was the oligo fluffy (expanded and dry) when you received it? If not, it may have taken on some moisture, which will make it very difficult to resuspend from this now hardened pellet. To avoid this, if the oligo is to be stored long term we suggest keeping the vial in a desiccator at room temp to 80C. Alternatively, you can aliquot the oligo right away and store the aliquots at 20 to 80C. If you are having difficulty resuspending an oligo, try heating to 65C for 10 minutes and vortexing. Leaving the oligo overnight on a vigorous shaker might also help dissolution. For difficult-to-dissolve oligos, try to make a stock no more copncentrated than 0.5 mM (i.e. 600 µL sterile water added to 300 nmol oligo).


• If the oligo was fluffy and you are still having problems resuspending it, it may be due to high G content or reduced solubility due to an added moiety such as our lissamine fluor. In these cases, again it is suggested that you heat to 65C for 10 minutes and vortex. Continue until completely resuspended. You can determine the concentration of your stock at any time following this protocol.
2. My oligo isn't working at all (no reduction of target protein)
• Do you have the correct concentration? Your stock oligo should be resuspended in sterile water without DEPC. You can always check the concentration of your oligo in the stock solution by following this protocol.


1. In the case of microinjection, make sure you are delivering the right concentration. The final concentration inside the embryo should be no less 2 µM. In most cases 2-10 ng injections are necessary to achieve good results.
2. For special delivery oligos, keep the Morpholino/DNA concentration at 1 µM and increase only the polyamine (EPEI) concentration to increase the level of oligo delivery.
3. For delivery-enabled oligos, make sure your final concentration is greater than or equal to 3 µM for optimal results.


• Do you have the correct target sequence? Make sure that the sequence of the oligo is the complement of a desirable target sequence. Keep in mind that inaccuracies in genbank file sequences or 'in-house' sequencing can contribute to improper targeting. Also, make sure that if the oligo is a translational blocker that the oligo targets sequence somewhere in the 5'UTR through the first 25 bases of coding sequence. If the oligo is a splice-blocking oligo then the sequence should target a region including a splice junction.


• Are you analyzing antisense activity at the right time? Make sure you are analyzing activity at the appropriate time. For example, a target mRNA encoding an enzyme may be shut down in 24 hours, whereas an mRNA encoding a ubiquitous structural protein may not be affected for days. Morpholino oligos are extremely stable and can be assayed a week after delivery as long as it is not diluted by cell division in the organism or cell culture. The decision as to how long to wait before assaying antisense activity is a judgment call to be made entirely by the researcher. However, this decision should be made based on the researchers knowledge of target specifics including, the target mRNA transcript concentration and stability, the protein concentration and stability, the potential for similar or redundant secondary targets, and the possibility of sequence anomalies or errors. Keep in mind that some highly expressed transcripts, like actin, may be difficult to shut down irrespective of oligo concentration.
3. My oligo works, but only reduces expression by 30% or less.
• In most cases simply delivering more oligo should increase the level of activity. However, keep in mind that specificity is inherently reduced as concentration is increased. For this reason it is important to deliver only enough oligo to achieve near-quantitative shutdown without affecting non-specific targets.


1. In the case of scrape-delivery or syringe loading, one can deliver as high as 50 µM without any detrimental effects on the cell or problems with target non-specificity.
2. For Special Delivery, there is no gain in exceeding the recommended 1 µM concentration described in the protocol as increasing the polyamine concentration becomes toxic and increasing the Morpholino/DNA heteroduplex concentration blocks the activity of the polyamine. The delivery-enabled Morpholino oligos will likely achieve better results as delivery is more efficient and greater concentrations can be used. Keep in mind that a subtle increase in concentration can change 30% shutdown to near-quantitative shutdown.


• There are other factors that can contribute to reduced activity which include potential secondary oligo targets and secondary structure within an oligo.


1. The secondary targets can come from homologous genes, particularly in the case of tetraploid organisms like Xenopus laevis. However it can also occur in non-tetraploid organisms when targeting one gene member of a family of homologous genes.
2. Secondary structure of an oligo can have a significant impact on activity. However, the concern is much greater for inter-strand pairing between oligos than intra-strand pairing within an oligo as inter-strand pairing can actually tie-up oligo thus eliminating it from the pool of oligo that can pair with target sequence. Either way, these concerns are typically not a problem, as oligos designed by Gene Tools should not exhibit significant intra-strand or inter-strand pairing.
4. My oligo used to work great and now it doesn't work anymore.
• Morpholino oligos are extremely stable. Oligos that have been resuspended in sterile, un-treated water and stored in aliquots at 80C should not lose activity. However, oligos can lose activity if improperly stored or stored in anything other than sterile pure water.


• It is possible for an oligo to lose activity if they have undergone long-term exposure to acid or have been exposed to DEPC, neither of which are present in oligos delivered to researchers. Gene Tools recommends resuspending oligos in sterile, un-treated water.


• It is possible for an oligo to fall out of solution if the stock concentration is close to it's solubility barrier and it has been frozen. Gene Tools highly recommends heating concentrated frozen stocks (1 µM or greater) at 65C for 10 minutes with vortexing prior to use. Continue this process until any particulates are dissolved. The concentration of your stock can be determined at any time by following our supplied protocol.