plate Probe size can range from about 20 nucleotides to the full- length template and longer(Moran et aL., 1996; Islas, Fairley, and Morgan, 1998). However, the bulk of the probe in most random prime labeling reactions is between 200 and 500nt If the entire 800 bp fragment is complementary to the intended target, a diverse probe population may not be detrimental. If only half the template contains sequence complementary to the target then sensitivity could be reduced. Any attempt to compensate by increasing probe concentration could result in higher back grounds. However, the major concern would be for the stringency of hybridization. Different wash conditions could be required to restore the stringency obtained with a probe sequence completely complementary to the target. 2kb DNA fragment The incorporation of radioactive label into probes generated by andom-primer labeling does not vary significantly between tem plates ranging from 300bp to 2kb, although the average size of probes generated from larger templates is greater(Ambion, Inc unpublished data). Generating a probe from a larger template could be advantageous if it contains target sequence absent from a smaller template The availability of different radioactive and nonradioactive labels could further complicate the situation, but the message re mains the same Visualize the hybridization event before you go to the lab. Consider the possible structures of your labeled probes and compare them to your target(s). Be prepared to change your labeling and hybridization strategies based on your experiments. duration, and resolution of the signal. One could aloc a Lab What Criteria Could You Consider When Selecting a Label One perspective for selecting a label is to compare the strength label's effect on incorporation into the probe, and the impact of the incorporated label on the hybridization of probe to target. The quantity of label incorporated into a probe can also affect the per formance of some labels and the probe's ability to bind its target Many experienced researchers will choose at least two techniques to empirically determine the best strategy to generate a new probe (if possible) Signal Strength and Resolution Signal strength of radioactive and nonradioactive labels is iversely proportional to signal resolution. Nucleic Acid Hybridization 405plate. Probe size can range from about 20 nucleotides to the full￾length template and longer (Moran et al., 1996; Islas, Fairley, and Morgan, 1998). However, the bulk of the probe in most random prime labeling reactions is between 200 and 500nt. If the entire 800bp fragment is complementary to the intended target, a diverse probe population may not be detrimental. If only half the template contains sequence complementary to the target, then sensitivity could be reduced. Any attempt to compensate by increasing probe concentration could result in higher back￾grounds. However, the major concern would be for the stringency of hybridization. Different wash conditions could be required to restore the stringency obtained with a probe sequence completely complementary to the target. 2kb DNA Fragment The incorporation of radioactive label into probes generated by random-primer labeling does not vary significantly between tem￾plates ranging from 300bp to 2kb, although the average size of probes generated from larger templates is greater (Ambion, Inc., unpublished data). Generating a probe from a larger template could be advantageous if it contains target sequence absent from a smaller template. The availability of different radioactive and nonradioactive labels could further complicate the situation, but the message re￾mains the same. Visualize the hybridization event before you go to the lab. Consider the possible structures of your labeled probes and compare them to your target(s). Be prepared to change your labeling and hybridization strategies based on your experiments. What Criteria Could You Consider When Selecting a Label? One perspective for selecting a label is to compare the strength, duration, and resolution of the signal. One could also consider the label’s effect on incorporation into the probe, and the impact of the incorporated label on the hybridization of probe to target.The quantity of label incorporated into a probe can also affect the per￾formance of some labels and the probe’s ability to bind its target. Many experienced researchers will choose at least two techniques to empirically determine the best strategy to generate a new probe (if possible). Signal Strength and Resolution Signal strength of radioactive and nonradioactive labels is inversely proportional to signal resolution. Nucleic Acid Hybridization 405