ULTRAhyb™-Oligo

ULTRAhyb-Oligo solution contains 25% formamide and is only compatible with positively charged or neutral nylon membranes.

The extreme sensitivity of ULTRAhyb-Oligo Hybridization Buffer may allow detection of RNAs that are not the expected full-length target. Although the probe binding could be legitimate (hybridization to alternatively spliced, partially degraded, or closely related mRNAs), some might be hybridization to RNAs with only partial homology to the target. The easiest way to reduce signal from cross hybridization (either cause) is to simply reduce the exposure time.
Here are other possible causes for cross-hybridization and solutions offered:
- Hybridization stringency may be inadequate. We recommend increasing the hybridization temperature by 2-5 degrees C and increasing the wash temperature by 5-10 degrees C.
- The probe contains non-target sequence. If the oligonucleotide has sequence homology with other mRNAs, vectors, etc., we recommend redesigning the probe to avoid sequence homology with targets other than the intended target.

Here are possible causes and solutions:
- Not enough probe was used or low specific activity probe was used. The amount and specific activity of probe needed to obtain radioactive signal above background depends largely on the amount of target on the blot and the specific activity of the labeled probe. Maximize signal by using a molar excess of probe labeled to the highest possible specific activity.

- Hybridization and/or washes are too stringent. If the oligonucleotide has a very low GC content or a low Tm, then lowering the hybridization temperature by 2-5 degrees C may increase the signal by reducing the hybridization stringency. Note that this may increase background and cross-hybridization. Alternatively washing can be done at room temperature instead of at 42 degrees C.

Here are possible causes and solutions:
- There could be precipitates in the ULTRAhyb-Oligo Hybridization Buffer. Inadequate solubilization of the hybridization buffer is one of the primary causes of high background. While warming the buffer at 68 degrees C, we recommend thoroughly mixing it with a gentle swirling motion. Ensure that there is no precipitate in the buffer before adding it to the blot. ULTRAhyb-Oligo Hybridization Buffer may start to precipitate at temperatures below 25-30 degrees C.

- Prehybridization may have been inadequate. We recommend increasing the blot prehybridization time from 30 min to 1 hr.

- Unincorporated radionucleotides may have been present. We recommend that free label be removed with a spin column (i.e., Ambion NucAway Spin Columns, Cat. No. AM10070) before adding the oligonucleotide probe to the hybridization solution.

- Washing may have been inadequate. We recommend the following:
-Ensure that your wash buffer contains SDS. Wash buffers lacking SDS are not recommended for use with ULTRAhyb-Oligo Hybridization Buffer.
-Do the post-hybridization washes in 5X SSC or SSPE, 0.5% SDS for 2 x 30 min. Increasing the salt concentration will help remove probe that is non-specifically bound to the membrane through electrostatic interactions.
-Double the post-hybridization wash time in 2X SSC or SSPE, 0.5% SDS from 2 x 30 min to 4 x 30 min.
-If none of the previous suggestions reduce non-specific background sufficiently, try increasing the post-hybridization wash temperature by 5-10 degrees C. This should be the last resort as it may remove hybridized probe in addition to reducing background.