PerfectHyb™ Plus

Product No. H7033
Technical Bulletin No. MB-610
Store at room temperature

Product Description

PerfectHyb™ Plus hybridization buffer has been optimized to yield maximum signal with minimum background in hybridizations as short as 1-2 hours. PerfectHyb™ Plus has been formulated to work in any hybridization protocol, utilizing any type of probe, and on any type of membrane (positively charged or neutral nylon and nitrocellulose).

Although signal continues to increase over time (see Figure 1), signal equivalent to overnight hybridizations using conventional hybridization buffers is achieved within 2-3 hours using PerfectHyb™ Plus. This allows the user to tailor the length of hybridization according to the needs of the experiment. For example, when conducting simple hybridization experiments such as screening PCR products by Southern blotting, a 30 to 60 minute hybridization is likely sufficient. If increased sensitivity is desired, the researcher can extend the hybridization to 2 to 3 hours to achieve signal equivalent to that observed in overnight hybridizations utilizing conventional hybridization buffers. To maximize sensitivity, the hybridization can be extended overnight to obtain a 2 to 5 fold increase in signal over conventional buffers without increasing background levels.

Many hybridization buffers, which yield increased rates of hybridization, are hampered by restrictions on the types or amounts of probe that can be used. PerfectHyb™ Plus yields high signal to noise ratios with both radioactive and non-radioactive labeled probes (see Figure 2). Any type of probe can be used including singlestranded or double-stranded DNA, RNA, and oligonucleotides. In addition, PerfectHyb™ Plus is not affected by the addition of excess or uncleaned probes.

Although PerfectHyb™ Plus will work in any hybridization protocol, the following procedures are recommended for maximum sensitivity.

Northern blot hybridizations comparing PerfectHyb Plus

Figure 1: Northern blot hybridizations comparing PerfectHyb™ Plus with a traditional hybridization buffer (0.25 M sodium phosphate, pH 7.2, 7% sodium dodecyl sulfate (SDS), and 2 mM EDTA). (A) Northern blots with 2 µg total mouse kidney RNA per lane were hybridized for the indicated amount of time with ~5 x 105 cpm 32P-labeled β-actin riboprobe per milliliter. After hybridization, blots were washed to high stringency, exposed and analyzed. (B) Counts per minute (cpm) obtained from blots shown in (A) plotted versus length of hybridization in hours.

Comparison of Northern blot hybridizations

Figure 2. Comparison of Northern blot hybridizations in different hybridization buffers for 1 and 20 hours. Northern blots containing ~2 μg human total RNA per lane were hybridized with ~200,000 cpm of 32P-labeled GAPDH RNA probe in the indicated hybridization buffer (Conventional buffer = 250 mM sodium phosphate buffer [pH 7.2], 7% SDS and 2mM EDTA). Blots were washed, imaged, and quantitated together on the Instant Imager.

Precautions and Disclaimer

PerfectHyb™ Plus is for laboratory use only, not for drug, household or other uses.

Storage/Stability

All materials may be stored at room temperature. Solutions have a shelf life of 1 year upon receipt. If a small amount of precipitate forms, heat the solution to 60-80 °C, mixing periodically, until dissolved. Once completely dissolved, invert to mix and store at room temperature.

Reagents that May be Required But are Not Provided

(Sigma product numbers are given where appropriate)

Preparation Instructions

Buffer Preparations

  • Low Stringency Wash Buffer (2X SSC, 0.1% SDS)
    To 500 ml molecular biology grade water, add 100 ml of 20X SSC stock solution and 10 ml of 10% SDS stock solution. Adjust volume to 1 L with water.

  • High Stringency Wash Buffer (0.5X SSC, 0.1% SDS)
    To 500 ml of molecular biology grade water, add 25 ml of 20X SSC stock solution and 10 ml of 10% SDS stock solution. Adjust volume to 1 L with water.

  • Ultra-High Stringency Wash Buffer (0.1X SSC, 0.1% SDS)
    To 500 ml of molecular biology grade water add 5 ml of 20X SSC stock solution and 10 ml of 10% SDS stock solution. Adjust volume to 1 L with water.

Procedure

Hybridization Procedure for Radioactive and Non-radioactive Probes

1.    Pre-hybridize membranes for at least 5 minutes at the appropriate hybridization temperature (see below).
       Longer pre-hybridizations are not necessary.

 

Probe Type Hybridization Temperaturea
DNA 68 °C
RNA 68 °C
Oligonucleotides 37-45 °C

a Temperatures for probes of irregular G+C content must be determined empirically.

Note 1: Sufficient volumes of hybridization solution must be used to ensure complete coverage of the membrane.

Note 2: Sigma has found that a blocking agent is not necessary when using PerfectHyb™ Plus. If a blocking
agent is preferred, the addition of single stranded DNA at 0.1 mg/ml is recommended.

 

Hybridization Vessel Recommended Volumes
Heat-sealable bags 100 μl/cm2
50 ml conical tubes 5 ml
Small Hyb tube 7 ml
Large Hyb tube 10 ml

 

2.    For double-stranded DNA probes, denature by heating probe to 100 °C for 10 minutes. Quick chill on ice for 2
       minutes. Single-stranded DNA and RNA probes do not require denaturation prior to addition to the hybridization
       reaction.


3.    Add denatured probe directly to pre-hybridization solution or add to fresh prewarmed PerfectHyb™ Plus and
       replace pre-hybridization solution with this mixture. For a radiolabeled probe, ≥1 x 106 cpm is usually sufficient
       to detect most targets.

4.    Choose the length of hybridization to achieve the required sensitivity according to the table below and the
       graph shown in Figure 1.

 

Blotting Application Required Sensitivity Recommended minimum
length of hybridization
Simple Southern blotb Low 30-60 minutes
Genomic Southern blot High 2-3 hours
Northern blot High 2-3 hours
Colony/Plaque lifts Low 30-60 minutes

b Blots screening plasmid preps or PCR products.

5.    After hybridization is complete, discard probe and wash blots once for 5 minutes at room temperature in low
       stringency wash buffer.

6.    For high stringency, wash twice for 20 minutes at hybridization temperature in high stringency wash buffer. For
       highest stringency, a final wash for 20 minutes at temperature with ultra-high stringency wash buffer may be
       added.

7.    If radioactive probes were used, the blots are now ready to be exposed to film. Wrap blots in plastic wrap or
       seal in bags. Expose to X-ray film using an intensifying screen at –70 °C.

8.    For non-radioactive probes, use colorimetric or chemiluminescent detection system according to manufacture’s
       instructions.

Troubleshooting Guide

Problem Cause Solution
Precipitate
present in
buffer
Storage temperature of buffer
falling below room temperature.
Heat to 60-80 °C for 15 minutes, mixing periodically.
Make sure the buffer is mixed well before use.
High
Background
Non-specific binding of probe to
target nucleic acids.
Add sheared, denatured salmon testis DNA (Product
No. D7656)
to a final concentration of 100 μg/ml in
prehybridization and hybridization solutions.
Wash conditions not sufficiently
stringent
Add the Ultra-High Stringency Wash step. Increase the
temperature of the hybridization and/or washes.
Exposure to film was too long Shorten the exposure time to film.
Concentration of enzyme
conjugate in non-radioactive
detection is too high.
Dilute the enzyme conjugate further. The specific dilution
required for optimal signal to noise must be determined
empirically.
Weak/Absent
Signal
Probe was not labeled efficiently Check that the specific activity of radiolabeled probes is
>5 x 108 cpm/μg. For non-radioactive probes, check the
incorporation of hapten by spotting and detecting serial
dilutions of probe in direct comparison to a known
standard. If probes are not labeled well enough, remake
and confirm adequate incorporation rates.
Target nucleic acids are not
present, have been degraded, or
are too low for detection.
Run agarose gel electrophoresis to confirm nucleic acids
are not degraded. Load more target nucleic acids for
blotting. For Southern blots, up to 10 μg DNA can be
loaded per lane. For Northern blots, up to 30 μg total
RNA can be loaded per lane.
Non-radioactive detection system
is not working properly.
Confirm the enzyme/antibody conjugate is functioning
properly by spotting and detecting the labeled probe on
nylon membrane. If the enzyme/antibody conjugate is
functional, check the chemiluminescent substrate by
spotting the enzyme/antibody conjugate on a membrane
and detecting with the substrate in question.

Materials


     

Reference

  • Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, (1989) second edition, Cold Spring Harbor Laboratory Press, New York

 

CDP-Star™ is a trademark of Tropix, Inc. Bedford, MA, USA

DAG/MAM 2/02

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