What is the sensitivity of the North2South Kit?
The North2South Kit is as sensitive as Southern and Northern blotting procedures using radioactive probes (32P or 35S). Femtomole quantities of biotin-labeled probe can be easily detected after binding to Streptavidin-HRP with only 5 to 10 minutes of exposure to X-ray film. The sensitivity of the assay will depend on the amount of label incorporation. For instance, a probe labeled with 10 molecules of biotin or HRP per strand will produce up to 10 times more signal than a probe with only 1 molecule of biotin or HRP per strand.
What length of probe should I use?
Nearly any length of probe can be used with this kit, as long as it has the required characteristics for specific hybridization and it has been labeled with biotin.
Will Biotin or HRP prevent the probe from hybridizing to the target sequence?
Biotinylated probes made by random incorporation (i.e, random prime labeling with biotinylated nucleotides) will have the biotin tag on the phosphate backbone of the probe. This will not affect the hybridization of nucleic acids to one another.
How can I biotinylate my probe?
Biotinylated DNA probes can be generated with the North2South Biotin Random Prime DNA Labeling Kit (# 17075). Probes may also be generated by PCR, random primed labeling or in vitro transcription. Biotinylated probes made with reagents and kits from other companies are compatible.
Can I use an end-labeled probe?
Short probes that are end-labeled with biotin may work with optimization to improve sensitivity. For longer probes, however, random-prime labeled produces better results because it adds multiple biotin group per molecule of probe.
How do I determine the sensitivity of my probe?
To determine probe sensitivity, perform serial dilutions of DNA that is complementary to the probe. The complementary DNA could be a purified plasmid, restriction fragment or PCR product containing the probe sequence. Separate the diluted DNA by electrophoresis and then transfer to a membrane or directly dot-blot the dilutions onto a membrane. Once the DNA is immobilized, UV crosslink it and perform the hybridization procedure with your labeled probe.
How long can I use a probe after labeling it?
Biotinylated probes can be stored at 4°C or -20°C indefinitely. The stability and labeling efficiency of the probe will be determined by the integrity of the nucleic acid and storage conditions.
Can I use the North2South Kit to perform colony lifts?
Yes. Standard colony lift protocols will work using a biotinylated probe. However, care should be taken to completely remove any residual bacterial debris or agarose from the filters, as this will cause high background signals. Non-specific speckling may be eliminated by centrifugation of the streptavidin-HRP conjugate in a bench-top microcentrifuge for several minutes at maximum speed before adding it to the hybridization buffer.
Can I use a CCD camera to record the results?
Yes. A CCD Camera will work with the North2South (#17097) Kit. The results will depend on the quality of the chip in the camera. The peak light emission for chemiluminescence is at 425 nm. Traditional phosphorescence imagers do not work for chemiluminescence.
Can I order the components of the North2South Kit separately?
Yes. Certain components are offered separately.
Is non-specific DNA added to the blocking buffer or hybridization buffers?
Yes. The hybridization buffers for the North2South Kit contain single stranded nucleic acids used to block the membrane during the pre-hybridization and hybridization procedures. The addition of more single stranded DNA or RNA is not needed.
Can I strip and re-probe a blot made used with the North2South Kit?
Yes. The most common procedure for stripping DNA blots is to wash the blot 3 x 5 minutes with boiling 0.1% SDS. An additional alkaline wash of 3 x 5 minutes with 0.4 N NaOH, 0.1% SDS may also work for DNA blots. The blots must be neutralized in TE buffer after this wash method. RNA blots are best washed with 0.1 % SDS, 3 x 5 minutes, at 75-85°C. Commercial stripping buffers are also available. Stripped blots should be kept wet until re-probed by storage in TE buffer at 4°C. Blots may be stripped and re-probed several times.