a) The expected lengths of the PCR product generated using the different primer combinations can be estimated by calculating the distance between the binding sites for the forward and the reverse primer (see Figure 2), e.g. the size of the PCR product using primer pair 8F-1492R is ~1500 ...
For larger or poorly amplified fragments, omit the dilution step. Note If multiple PCR products are observed on the gel, or when cloning very large PCR products, gel isolate the desired PCR product prior to performing the ligation reaction. See Appendix I for a gel-isolation protocol. For a...
While Squash-PCR is recognized as an easy and reliable method, further improvements are necessary to tackle larger-scale projects and process a substantial number of samples with ease. Automation of the squashing process can streamline sample preparation and increase the number of samples processed si...
When PCR reaction Eight individual loci are amplified with similar intensities when the primer pairs are used separately. When equimolar amount of these primers are mixed together for a multiplex reaction (Mix K), some of the products are much weaker (#1, #2, #5, #6) than other. In this...
It is likely that this larger fragment car- ries a WT PMS1 gene. Note that none of the pms1:TRP1 candidates that re- tained a WT PMS1 allele yielded the 2.1-kbp PCR product expected from this allele (Figure 2). This is probably a result of a more efficient amplification of the ...
Usually the optimal annealing temperature is 5°C lower than the melting temperature of primer-template DNA duplex. Incubation for 0.5-2min is usually sufficient. However, if nonspecific PCR products are obtained in addition to the expected product, the annealing temperature should be optimized by in...
All assay showed ΔCq larger than 10 between RT and RT(−) control evidencing negligible DNA background. Inhibition in the RT-qPCR workflow was tested using an RNA spike (TATAA Biocenter) and no inhibition was detected. 4.2. Preparation of standard curves A pipetting robot EP Motion EP5070...
PCR product from genomic contamination that will be larger in size than the product generated from the cDNA. In fact, primers can be designed to span a sufficiently large genomic fragment such that amplification from contaminating DNA may be not be possible. For genes in which the genomic ...
Usually the optimal annealing temperature is 5°C lower than the melting temperature of primer-template DNA duplex. Incubation for 0.5-2min is usually sufficient. However, if nonspecific PCR products are obtained in addition to the expected product, the annealing temperature should be optimized by in...
For reasons that are not fully understood, this results in the res- olution of DNA fragments much larger than can be resolved by conventional agarose electrophoresis. The early versions of this method made use of a field that period- ically reversed its direction. This was termed pulsed-field...