Many people suggest that the DNA probe for Northern needs to be minimum of 300bp long. Is there a upper length limit?
I am trying to troubleshoot Northern using plant RNA and many people are scared with northern in plants. I am planning to use Rediprime II DNA Labeling System for labeling. Considering this, is there a upper limit for the length of a probe?
Most important is that your probe is specific enough for your target gene. The longer the probe, the more likely you will have homology with additional genes. Sure, you can increase the stringency for the hyb and washes, but it starts with highly specific probe. In theory there is no upper limit given probe is specific. Smaller probes than 300bp also work fine, but labelling efficiency (density) should be sufficient. That is why most people prefer probes >300bp.
For Northern blot probe, the specificity is the most important. Before you select the probe, you should analyze its specificity by blast program. Of course, you can improve the specific by more stringent hybrid and washing condition. As for the size of probe, I think that it theoretically is no limit. Considering the labelling efficiency of probe, you should more than 300 bp in size.
Thank you so much Drs. van der Graaff and Li for your suggestions. I am trying to design my probes with ca. 500bp and checking them via blast. I will update the Research Gate community my results.
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