Technical Tips: 40 mer Probes
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1. What type of specific activity is needed in radiolabeled probes?Top
For best results, it is critical that the probe has a high enough specific activity. For 32P end labeling, the specific activity should be at least 3 x 106 cpm/pmol or 3 x 108 cpm/g. If you do not get a high enough specific activity, it is usually a problem with the enzyme. Check to be sure the reaction buffers are correct and, if necessary, purchase new enzyme from a high quality supplier.
2. Is pretreatment needed?Top
Spin the vial before use. This will prevent losing probe that may have become "trapped" is on the cap of the vial. No treatment with alkaline phosphatase is needed. These probes are chemically synthesized and do not have a phosphate group on the end (both ends are -OH). It is not necessary to boil the probe, as this is a single-stranded probe.
3. How do I label the probe?Top
Do not use random priming or nick translation to label these probes because they are too short; use end-labeling. You can label with biotin or digoxigenin. Using commerically available kits, you can concentrate the probe (i.e. with a speed vac) to get the recommended volume and label the entire vial of probe. Store the labeled probe and use as needed. Do not use kits that requires an amino group at the 5 end; this is not present on our probes (both ends are -OH).
4. Can I use the probes for Northern blots?Top
For Northern blots, use polyA RNA, not total RNA. PolyA RNA will give a 10X stronger signal. Total RNA may give background bands at 18S and 28S corresponding to ribosomal RNA. For Northern blots, we use the same hybridization conditions as described for Southern blots. Since every RNA preparation has a different mix of RNAs, you may want to optimize the hybridization conditions for your sample.
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