PRODUCT: HIGH EFFICIENCY HYBRIDIZATION SYSTEM - SUPER HYB® February 2006

Cat. No. HS 114
               HS 114F (with 50% formamide)
Storage: Store at room temperature. Use within two years from the date of purchase.

PRODUCT DESCRIPTION

The High Efficiency Hybridization System - Super Hyb Solution 7, cat. no. HS 114 (F), is a ready to use hybridization solution for northern, Southern and microarray hybridization. The HS 114 (F) has been formulated to provide the highest hybridization efficiency available, combined with low background. The exceptional performance of the HS 114 (F) allows detection of low abundance mRNA in preparations of total RNA, thus eliminating the need for isolation of a poly A⁺ fraction. HS 114 (F) may be used for radioactive or non-radioactive detection. HS 114 (F) can be purchased separately or as a part of the Super Hyb Kit, cat. no. SK 116
The composition of HS 114 (F) includes dextran sulphate for the enhancement of hybridization signal and Background QuencherJ for blocking of non-specific binding of hybridization probes. HS 114 contains no formamide and 114F is supplemented with 50% formamide. The hybridization signal enhancement in HS 114 (F) relies upon formation of a dextran sulphate supported network of a DNA probe. In effect, several molecules of the probe can be attached to a single molecule of the membrane-bound RNA or DNA. Because of the signal enhancement, the double-stranded DNA probes perform better than single-stranded DNA or RNA probes.

APPLICATION

1. PREHYBRIDIZATION (Optional)

Prehybridization is not required for hybridization performed with HS 114 (F) in a rotating tube. If hybridization is performed in a plastic bag, the following prehybridization protocol can be applied.
Dilute 10X Washing and Prehybridization Solution (cat. no. WP 117) to obtain a 1X solution. Alternatively, prepare a prehybridization solution by mixing 0.58g NaCl and 1g SDS with 99 ml of water (0.1 M NaCl and 1% SDS). Supplementation of the prehybridization solution with salmon sperm DNA or RNA is not required. Place the hybridization membrane in a hybridization bag and add sufficient amount of the prehybridization solution to wet the membrane. Carry on prehybridization for at least 20 minutes at room temperature.

2. HYBRIDIZATION

Denature the double stranded DNA probe by boiling in 1 mM NaOH for 5 - 10 min. For optimal hybridization, use a sufficient amount of the probe to approach 1 - 2 x 10⁶ dpm per ml of hybridization solution. For ³²P probes older than 6 days, increase the amount of radioactivity to 5 x 10⁶ dpm per ml of hybridization solution. ³²P probes can be used for hybridization up to 14 days following labeling, but with decreased hybridization efficiency. For non-radioactive probes, use 5 - 20 ng of probe per ml of hybridization solution.
Rotating tubes. Pour at least 5-10 ml of HS 114 (F) into a tube containing hybridization membrane. Use 60-100 µl of HS 114 (F) per cm² of the membrane. Wet the membrane completely with the solution. Keep the tube vertical and add the denatured probe to the hybridization solution at the bottom of the tube. Mix the contents and place the tube in a hybridization oven.
Plastic bags. Remove the prehybridization solution by rolling a pipet over the hybridization bag. Add the denatured probe to a tube containing the HS 114 (F) solution (60 - 100 µl per cm² of the hybridization membrane). Mix the contents and place the open hybridization bag with the membrane over the top of the tube with the HS 114 (F) - probe solution and invert the tube and bag, allowing the radioactive hybridization solution to be securely transferred into the hybridization bag.
Micorarrays. HS 114 (F) provides excellent results with microarray hybridization using glass or plastic based matrixes. Protocols for microarray hybridization may vary. The following is a general guideline for the use of HS 114 (F).

Perform prehybridization with WP 117 for at least 20 minutes. For expression profiling, supplementation of WP 117 with 0.5 µg/ml DNA and 0.5 µg /ml oligo (dA) might be required. At the end of prehybridization, warm the HS 114 (F) and hybridization chamber to appropriate hybridization temperature (42 or 65 C). Add the denatured probe to pre-warmed HS 114 (F) and add the probe-HS 114 (F) solution to a hybridization chamber.

DNA hybridization. Carry on hybridization for at least 16 h at 65 C using HS 114 or at 42 C using HS 114F (containing 50% formamide).
RNA hybridization. Carry on hybridization for at least 16 h at 62 C using HS 114 or at 42 C using HS 114F (containing 50% formamide).It is safe to extend hybridization time up to 72 h, thus allowing hybridization to be performed over the weekend.

3. WASHING

HS 114 (F) efficiently blocks non-specific hybridization, allowing all washes to be performed at room temperature. (See Note 4). Following hybridization, dilute the 10X Washing and Prehybridization Solution ( WP 117) to obtain a 1X solution. Place the membrane in a tray with 1X Washing and Prehybridization Solution. Alternatively, use washing solution containing 0.3 M NaCl and 30 mM sodium citrate (2X SSC) supplemented with 1% SDS. The membrane should float freely in the washing solution. Place the tray on a shaker and shake the membrane for 7-10 minutes at room temperature. Pour off the washing solution and repeat the washing cycle five times.
Shaking (or rolling) is critical for the efficient removal of a probe from the non-specific binding sites on a membrane. For most hybridization probes, washing at room temperature provides optimal results. For probes yielding high background, perform the last two washes at 50 C in a shaking water bath or in the hybridization oven with the rolling tubes.

After washing, wrap the wet membrane in Saran Wrap or Glad Wrap for autoradiography. For non-radioactive detection procedures proceed with blocking and binding steps. Keeping the membrane wet during detection procedures is critical for probe removal if the membrane will be used for re-hybridization.

4. PROBE REMOVAL FROM NYLON MEMBRANES

Place the membrane in a plastic bag partially filled with 1X Washing and Prehybridization Solution. Alternatively, use a solution containing 1% SDS and 40 mM Tris-HCl (pH 7.5 - 7.8). Place the bag in boiling water for 5 minutes. Pour out the solution and repeat this washing process 3 - 4 times.

NOTES

1. We have developed effective procedures for the downward transfer of RNA and DNA from agarose gels to hybridization membranes. By reversing the flow of transfer solution from upward to downward, the downward transfer protocols shorten the RNA and DNA transfer to 1 hour, are easy to set up, and result in increased signal sensitivity. See references 1 and 2 for details.
2. Addition of salmon sperm DNA to HS 114 (F) is not necessary. HS 114 (F) contains Background Quencher^TM which efficiently blocks non-specific binding of hybridization probes. Background Quencher^TM does not contain nucleic acids. This is especially advantageous for hybridization with probes containing conserved sequences since nucleic acids added to a hybridization solution may contain homologous sequences interfering with the probe hybridization.
3. ³²P-labeled probes. Label the double stranded DNA probe by nick-translation or primer extension method. To prevent radioactive contamination, perform labeling in screw cap microcentrifuge tubes and avoid the use of snap top tubes. For the highest hybridization sensitivity, the probe specific activity should be about 10⁹ dpm/g DNA. When possible, use cDNA inserts rather than the whole vector containing the insert and plasmid DNA. To avoid non-specific binding, DNA probes should not be contaminated with bacterial genomic DNA.

The following nick-translation protocol consistently yields probes with specific activity > 10⁹ dpm /µg DNA (using either LTI or Amersham nick-translation kits). To prepare a reaction mix, keep the reaction tube on ice and add ingredients in the following order:

1. DNA, 50 - 100 ng 1 µl
2. 10X buffer 2.5 µl
3. ³²P CTP (3000 mCi/mmol) 19 µl
4. 10X enzyme solution 2.5 µl

Mix the ingredients and carry on nick-translation for 1 hour at 14 C. To label 10-25 ng of DNA, the reaction can be scaled down to 12.5 µl.
Following labeling, add to the reaction tube: 5 l of Polyacryl Carrier (cat. no. PC 152) or 5-10 g of carrier DNA or RNA. Supplement the mixture with 5 µl of 4 M NaCl. Mix the tube contents and add 1 ml of 80% ethanol. Vortex the solution, store for 2-3 minutes at room temperature and sediment the precipitated probe at 10,000 g for 5 minutes at 4-25 C. No additional purification of the radioactive probe is necessary. Dissolve the probe in 1 mM NaOH and store at 4 C. The membrane wash at elevated temperature is not necessary when using HS 114 or HS 114F.

Both solutions effectively reduce binding of the probe to the hybridization membrane and minimize mismatches of the probe with non-target RNA or DNA sequences. A similar phenomenon was observed recently (3).

REFERENCES

1. Chomczynski P and Mackey K (1994) One-hour downward capillary blotting of RNA at neutral pH. Anal. Biochem. 221, 303-305.
2. Chomczynski P (1992) One-hour downward alkaline capillary transfer for blotting of DNA and RNA. Anal. Biochem. 201, 134-139.
3. Cornish EC, Beckham SA and Maddox JF (1998) Southern hybridization revisited. Biotechniques 25, 947-953.

High Efficiency Hybridization System - Super Hyb®, Manufacturer protocol, 2004.
Super Hyb® is registered trademark of Molecular Research Center, Inc.