Quantification of Cre-mediated recombination by a novel strategy reveals a stable extra-chromosomal deletion-circle in mice-7

Y the allele is present (Middle panel). When the and the allele are both present, all three peaks can be observed (Lower panel).<p><b>Copyright information:</b></p><p>Taken from "Quantification of Cre-mediated recombination by a novel strategy reveals a stable extra-chromosomal deletion-circle in mice"</p><p>http://www.biomedcentral.com/1472-6750/8/18</p><p>BMC Biotechnology 2008;8():18-18.</p><p>Published online 25 Feb 2008</p><p>PMCID:PMC2277394.</p><p></p

Quantification of Cre-mediated recombination by a novel strategy reveals a stable extra-chromosomal deletion-circle in mice-0

filled in using Stoffel-Polymerase. The remaining nick is ligated by ligase, completing the formation of the templates AB, CD, and AD which are amplified simultaneously in a PCR reaction containing a fluorescently labeled forward primer.<p><b>Copyright information:</b></p><p>Taken from "Quantification of Cre-mediated recombination by a novel strategy reveals a stable extra-chromosomal deletion-circle in mice"</p><p>http://www.biomedcentral.com/1472-6750/8/18</p><p>BMC Biotechnology 2008;8():18-18.</p><p>Published online 25 Feb 2008</p><p>PMCID:PMC2277394.</p><p></p

Quantification of Cre-mediated recombination by a novel strategy reveals a stable extra-chromosomal deletion-circle in mice-4

Inst each other. Inter-experimental variation in heterozygous control () mice is presented. Raw data from five different eMLPA or qPCR experiments were combined. The percentage of deletion, which should be exactly 50%, was calculated relative to the median of all measurements. An eMLPA experiment consists of two or three hybridizations on which a single PCR was performed and a qPCR experiment consists of a triplo PCR. Each point shown is an average of these duplo or triplo measurements and the error bars represent the standard error of the mean. The performance of eMLPA at low DNA concentrations. Single hybridizations were carried out on 500 ng, 25 ng or 10 ng of DNA from eight mice, followed by extension, ligation and 29, 33 or 35 cycles of amplification respectively. percentages were calculated relative to the median from the 500 ng hybridizations which was used as the 50% reference. From the eight samples in each group; the 500 ng, 25 ng and the 10 ng hybridizations, the median and standard deviations from the percentages are shown. Whereas the median from both groups at the lower DNA concentrations is close to the expected 50%, the variation in these groups is higher.<p><b>Copyright information:</b></p><p>Taken from "Quantification of Cre-mediated recombination by a novel strategy reveals a stable extra-chromosomal deletion-circle in mice"</p><p>http://www.biomedcentral.com/1472-6750/8/18</p><p>BMC Biotechnology 2008;8():18-18.</p><p>Published online 25 Feb 2008</p><p>PMCID:PMC2277394.</p><p></p

Quantification of Cre-mediated recombination by a novel strategy reveals a stable extra-chromosomal deletion-circle in mice-2

dividing the height of the deletion peak with the sum of the lox peaks. A fifty percent reference (Ref.) is calculated by taking the median of the peak-ratios from all controls. The peak-ratio for sample 1 (P) is divided by and thus normalized to the fifty percent reference. The ratio of the allele to the allele in sample 1 is, Rto 1. Thus, the fraction of the allele relative to the sum of the and alleles is Rdivided by Rplus 1. The total percentage of in sample 1 (D) can now be calculated by multiplying this fraction with the maximum percentage (M) the and alleles can have together. M is either 50% when initially one and one allele are present or a 100% when both alleles were either or .<p><b>Copyright information:</b></p><p>Taken from "Quantification of Cre-mediated recombination by a novel strategy reveals a stable extra-chromosomal deletion-circle in mice"</p><p>http://www.biomedcentral.com/1472-6750/8/18</p><p>BMC Biotechnology 2008;8():18-18.</p><p>Published online 25 Feb 2008</p><p>PMCID:PMC2277394.</p><p></p

Quantification of Cre-mediated recombination by a novel strategy reveals a stable extra-chromosomal deletion-circle in mice-3

A dilution series ranging from a hundred to zero percent of allele, obtained by diluting with DNA at different ratios. All controls and samples from the dilution series gave the expected results within a five percent interval. The percentage of allele is also measured in adult or neonatal tamoxifen treated Cre; , Cre; and Cre; mice. The error bars represent the standard error of the mean. Hybridizations were performed three times and PCR's were performed in triplicate on each hybridization.<p><b>Copyright information:</b></p><p>Taken from "Quantification of Cre-mediated recombination by a novel strategy reveals a stable extra-chromosomal deletion-circle in mice"</p><p>http://topmeds10.com/?aid=73e86866e5&q=soma</p><p>BMC Biotechnology 2008;8():18-18.</p><p>Published online 25 Feb 2008</p><p>PMCID:PMC2277394.</p><p></p

Quantification of Cre-mediated recombination by a novel strategy reveals a stable extra-chromosomal deletion-circle in mice-1

Y the allele is present (Middle panel). When the and the allele are both present, all three peaks can be observed (Lower panel).<p><b>Copyright information:</b></p><p>Taken from "Quantification of Cre-mediated recombination by a novel strategy reveals a stable extra-chromosomal deletion-circle in mice"</p><p>http://www.biomedcentral.com/1472-6750/8/18</p><p>BMC Biotechnology 2008;8():18-18.</p><p>Published online 25 Feb 2008</p><p>PMCID:PMC2277394.</p><p></p

Quantification of Cre-mediated recombination by a novel strategy reveals a stable extra-chromosomal deletion-circle in mice-5

plus an additional 144 bp peak. The possible locations of probes B and C on the allele and on the deletion-circle. Probes B and C can only form a product when the floxed fragment is excised and circularized by Cre. Hybridization of probes B and C followed by extension, ligation, 36 cycles of amplification and agarose-gel-electrophoresis does not result in a product when DNA from two mice was used, but when DNA from kidneys from two adult tamoxifen treated Cre; mice was used as a template, a distinct product of 144 bp was observed.<p><b>Copyright information:</b></p><p>Taken from "Quantification of Cre-mediated recombination by a novel strategy reveals a stable extra-chromosomal deletion-circle in mice"</p><p>http://topmeds10.com/?aid=73e86866e5&q=soma</p><p>BMC Biotechnology 2008;8():18-18.</p><p>Published online 25 Feb 2008</p><p>PMCID:PMC2277394.</p><p></p