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The following chapter describes the options how to label nucleic acids with DIG. Enzymatic labeling procedures Principally all enzymatic labeling techniques can be applied with our non-radioactive reagents. Here we will only discuss the most important ones: Random Primed Labeling using Klenow PCR, using Taq polymerase, Pwo polymerase or Expand HiHi or Long Range dNTPack Oligonucleotide 3-endlabeling or tailing, using Terminal Transferase RNA-labeling during in vitro transcription, using SP6, T3 or T7 RNA polymerase Nick Tranlation which is mainly used for in situ applications will not be discussed 用一些六核苷酸作为随机引物，将这些引物和探针DNA片段一起热变性，退火后，引物与单链DNA互补结合，再在DNA聚合酶的作用下，按碱基互补配对原则不断在其3`- OH端添加标记的单核苷酸修补缺口，合成新的标记的探针片段。 For RPL a very pure template DNA is required The template is denatured in a boiling water bath, cooled on ice and then the labeling components are added.(Klenow polymerase. - buffer, DIGdUTP/TTP mixture, hexamer primers). The mixture of DIGdUPT:TTP here is 1:3. It is very important that the DIGdUPTs are incorporated with a certain spacing so that only every 20th – 25th nucleotide contains a DIG molecule and this is achieved by using a ratio of approximately 1DIGdUTP plus 2 TTPs). DIG labeled DNA probes are generated at high yield within one hour or overnight. We provide a Standard RPL Kit and “DIG HIGH Prime” which is an improved version, resulting in more probe per ng of template compared to the Standard RPL Kit. Random primed labeling reactions can be scaled up, if more probe is needed. This may be of special interest to diagnostic labs which are using the same probe-sequence for their tests over and over again. They could label mg amounts of template with DIG, evaluate the labeling efficiency once and use this probe for the next years. The stability of DIG-labeled probes, as we now know, exceeds our warranty of 1 year by far. RPL is somewhat outdated. The easier way to label probes successfully is by PCR. We offer an optimized version of the st