( lified using aspecific set of primer...)High resolution DNA analysis with the,,,DNA 500 and DNA 1000 LabChip kits

lified using a specific set of primers to result in a 439 bp fragment for the amplification of GAPDH and a 650 bp fragment for the amplification of hsp72. A primer dropping method was employed because the two genes are expressed at different levels within the cell. In the present case this means that hsp72 was amplified for 40 cycles and the primers for the amplification of GAPDH were added at the beginning, after 2 cycles, 4 cycles and so on, resulting in 20 to 40 amplification cycles. Since the PCR reaction was carried out under competitive conditions, the PCR product of hsp72, though at a constant amplification of 40 cycles, disappears with increasing cycle number of GAPDH (figure 3).

Figure 3 shows the separation of the two PCR products as a gellike image and also shows a plot of the concentrations that were calculated by the bioanalyzer for the different amplification conditions. From the results it was concluded that a primer dropping method with 40 cycles for hsp72 and 36 cycles GAPDH would be preferable. Under these conditions a large GAPDH and a small hsp72 peak are visible. This allows monitoring up-regulation of the hsp72 gene in future experiments. For other experiments, a different number of cycles might be preferable.
The advantage of using the DNA 1000 assay for the analysis of competitive PCR products lies in the accurate absolute and relative quantitation of each amplified product. Small differences in the amplified amount, that cannot be detected using slab gel analysis, are easily analyzed with the DNA 1000 assay. This allows not only the optimization of PCR conditions but also detection of changes in gene expression via RT-PCR methods.
Conclusion
The DNA 500 and DNA 1000 assay perform DNA separations reliably in the range
'"/>

Source:


Page: All 1 2 3 4 5
Related biology technology :

1. Analysis of urine and seawater samples by ultrasonic nebulization with a high resolution ICP spectrometer
2. High-throughput gene expression analysis to screen for anti-asthma drugs
3. Fully automated DNA purification and efficient multiplex PCR for analysis of microsatellite loci
4. Flow cytometric analysis of human primary cells using the Agilent 2100 bioanalyzer and on-chip staining
5. 2100 expert software Powerful software for the analysis of RNA, DNA, proteins, and cells with the Agilent 2100 bioanalyzer
6. Using the Agilent 2100 bioanalyzer for quality control of protein samples prior to MS-analysis
7. Microfluidic analysis of multiplex PCR products for the genotyping of Helicobacter pylori
8. Extended data analysis using DeCyder EDA
9. 2D-LC analysis of phosphopeptides in brain tissue using Ettan MDLC and Finnigan LTQ
10. Neurite outgrowth cell-by-cell analysis using the IN Cell Developer Toolbox
11. Laser Capture Microdissection of muscle fiber populations and expression analysis by RT-PCR