By Detlef Weigel; Jane Glazebrook
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ACÁ TCT AC 3' Rßorder: 5' TGG GAA AAC CTG GCG TTA CCC AAC TTA AT V Better results are obtained if the primers arc lJAGK-purified. If a known insertion is present in the pools that arc being screened, it is a good idea to try to detect that insertion before searching for an insertion in YFG. Such a positive control ensures that the PCR conditions arc correct. 2. Design primers for YFG. One primer reading in from the 5' end of the coding sequence and one primer reading in from the 3' end are needed.
Hanhart C , Soppe W , and Peeters T. 1994. The phenotype of some late-flowering mutants is enhanced by a locus on chromosome 5 that is not effective in the Landsberg erecta phenotype. Plant J. 6: 911-919. R. 1999. T-DNA as an insertional mutagen in Arabidopsis. Plant Cell 11: 2283-2290. , and Amasino R. 1993. Analysis of naturally occurring late flowering in Arabidopsis thaliana. Mol. Gen. Genet. 237: 171-176. McCallum C M . , and Henikoff S. 2000. Targeted screening for induced mutations. Nat.
If no positive signals are obtained, and experimental error has been ruled out, the conclusion must be that there are no insertions in YFG in this collection. The process must start again using a different collection. The probability of finding an insertion in YFG in a population of a certain size can be calculated from the Poisson distribution, assuming that the distribution of insertions is random. For example, let us say that the coding sequence of the gene of interest is 2 kh. 0000159. Say that the population being screened contains 60,000 different insertions.
Arabidopsis : a laboratory manual by Detlef Weigel; Jane Glazebrook