Bio-Rad Zeta-Probe Membranes - Manual (Page 8)

Alternative hybridization protocols are necessary when probe lengths
vary outside this recommended range (refer to Oligonucleotide
Protocol, Section 4.4).
Template purity is essential during probe synthesis, especially probes
made by random primer extension. Small amounts of contaminating
DNA templates can cause lane background or extra bands due to the
high specific activity of random priming.
Optimal probe specific activity and concentration can vary according
to available hybridization sites and exposure time. Probe cleanup is
essential to minimize background. Unincorporated nucleotides pre-
sent after probe preparation contribute to hybridization background.
The most effective cleanup method is desalting by column separation.
This can be done in a column (1 to 5 ml bed volume) using Bio-Gel
®
P-30 gel (catalog #150-1340) or with Bio-Spin ® 30 columns (catalog
#732-6004).
After cleanup, denature the double-stranded probe by increasing
temperature to 95–100°C for 5 min. Then cool rapidly on ice. Use the
probe as soon as possible after preparation.
Section 4
Hybridization Protocols for DNA Probes
There are several hybridization protocols that will give high quality
results. The key to successful nucleic acid blotting is proper blocking of
the Zeta-Probe membrane. The most important blocking reagent in the
hybridization solution is sodium dodecylsulfate (SDS). SDS is most
effective when used at concentrations ³1% (w/v). The Standard
Protocol (Section 4.1) uses 7% (w/v) SDS, which has been shown to
give extremely low background and high signals. The protocol
described in Section 4.2 includes formamide, which allows
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8. Disconnect the vacuum. Remove the blotted Zeta-Probe
membrane.
9. Remove the glyoxal by rinsing the membrane in 2x SSC and
letting it airdry. Fix RNA onto Zeta-Probe membrane by
baking the membrane at 80°C for 1 hr.
2.7 DNA Alkaline Fixation
After transfer, place the Zeta-Probe membrane (DNA surface facing
up) on a pad of 3MM paper saturated with 0.4 M NaOH for 10 min.
Rinse in 2x SSC and air dry. The dried membranes are stable at room
temperature. The membranes can be stored between two pieces of
filter paper in plastic bags at 23–25°C.
Section 3
Probe Recommendations
The specific activity, concentration, size range, and purity of the probe
all have an important effect on signal-to-noise ratio during hybridiza-
tion. For hybridization on Zeta-Probe blotting membranes, the
following is recommended:
Probe specific activity 10 8 cpm/µg probe
Probe concentration in
the hybridization mixture
10 6 cpm/ml (10–50 ng/ml)
Probe length 200–1,000 bp
Probe length is an important parameter to control. DNA probes
prepared by random priming tend to be small. Small probes can
cause lane specific background during low stringency hybridizations.
DNA probes prepared by nick translation are generally long. Probe
fragments longer than 1 kb decrease hybridization specificity.
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