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The PicoPure DNA Extraction Kit provides conveniently packaged, stable Proteinase K, PCR-compatible DNA Reconstitution Buffer and a complete user guide. In this reformatted Kit, each of ten lyophilized Proteinase K aliquots can be freshly reconstituted to make 150 µl of PicoPure DNA Extraction Solution-enough for 150 x 10 µl CapSure HS LCM Cap extractions or 30 x 50 µl CapSure Macro LCM Cap extractions-all at a much lower price.
Reproducible DNA extraction and PCR amplification of multiple single-copy genes from ten Laser Capture Microdissected cells. Cells were microdissected from human peripheral white blood cell cytospins, and DNA was extracted using 10 µl of PicoPure DNA Extraction Solution. The extracts were analyzed directly in 35-cycle PCRs using primers for three single-copy genes. M: DNA Marker (Sigma). Lane 1-5: 536 bp human b-globin gene fragment. Lanes 6-10: 245 bp cystic fibrosis gene fragment (CFTR). Lanes 11-15: 125 bp human p53 gene fragment. Lane 16: No template, negative control PCR.
Maximaze DNA Recovery
Because DNA is not lost due to organic extractions or spin columns, the PicoPure Kit enables reliable and reproducible DNA recovery from as few as ten cells.
PCR studies illustrate the advantages of the PicoPure Kit. Equal amounts of DNA were transferred to PCR, either directly in the PicoPure Kit Buffer or after purification using a DNA Extraction Kit from a leading manufacturer. Quantitative Real-time PCR (QRT-PCR) demonstrates that the PicoPure Kit samples amplify earlier, suggesting more DNA present, and amplify more consistently, indicating less sample variation, than column-purified samples.
The PicoPure Kit's reliably high DNA recovery enables increased detection sensitivity, for example, when amplifying single-copy genes from small samples in mutation analysis or genotyping assays. This high DNA recovery makes the Kit ideal for small microdissected samples, such as those prepared by LCM.
Superior DNA recovery using the PicoPure DNA Extraction Kit. Five, 1 ng replicates of DNA were diluted in 10 µl PicoPure DNA Extraction Buffer, or using the other manufacturer's kit, five, 1 ng replicates of DNA were column purified and concentrated to 10 µl. A human beta-globin gene fragment was then amplified from the samples by Real-Time PCR (40 cycles). Cycle thresholds for the PicoPure Kit samples were 28.9 ± 0.28 and 32.4 ± 1.35 for the column-purified samples, respectively.
Extract DNA For Any PCR Application
DNA extracted with the PicoPure Kit is compatible with endpoint PCR-performed in the same 0.5 ml tube used for DNA extraction. For gene copy number quantitation, DNA from very small samples can be directly amplified by QRT-PCR without further purification, using platforms such as the LightCyler® System (Roche). For highly sensitive mutation and genotype analysis, DNA can be extracted and directly amplified from small samples, and gene fragments can be separated by dHPLC (denaturing High-Performance Liquid Chromatography), using platforms such as the WAVE® System (Transgenomics).
PicoPure DNA extraction and quantitation of Her2/neu gene copy number by Real-Time PCR. 10, 100, and 1000 SKBR3 cells were microdissected from cell smears using LCM. DNA was extracted using 10 µl of PicoPure DNA Extraction Solution and, without further purification, all 10 µl was used in a 20 µl LightCycler® reaction. The number of gene copies per sample was determined and divided by the number of cells per sample to determine the number of gene copies per cell.
PicoPure DNA Extraction Solution is compatible with endpoint PCR and amplicon detection using dHPLC. DNA was extracted from 10 peripheral white blood cells per sample using the PicoPure DNA Extraction Kit. This DNA was used, without further purification, in PCRs to amplify a 125 bp human p53 gene fragment and a 245 bp human cystic fibrosis gene (CFTR) fragment. The PCR products were then separated and detected using the dHPLC WAVE® System. Data courtesy of Scott Hamlin and Nicolas Neckelmann, Transgenomics, Inc., San Diego, CA.
Use With Most Tissue And Cell Preparation Procesures
The PicoPure DNA Extraction Kit enables successful recovery of genomic DNA from animal tissue section and cell samples prepared using a wide range of methods1. Superior results are obtained from paraffin-embedded formalin-fixed tissue sections, frozen tissue sections, ethanol fixed cells, and cytological smears.
1Not recommended for use with whole blood, plant or fungal samples.
PicoPure DNA extraction and b-globin gene amplification from LCM samples prepared by various methods. Cell and tissue samples were prepared for LCM using the methods listed below. LCM was used to collect samples. DNA was extracted using the PicoPure DNA Extraction Kit, a single-copy 536 bp human b-globin fragment was amplified from each sample using standard protocols, and the products were electrophoresed on a 10% polyacrlamide/TBE gel and stained with SYBR® Gold Nucleic Acid Stain (Molecular Probes). M: DNA marker. Lanes 1-3: ethanol-fixed SKBR3 cells. Lanes 4-6: formalin-fixed, paraffin-embedded breast tissue. Lanes 7-9: frozen, ethanol-fixed foreskin tissue. Lane 10: positive PCR control of 1 ng human genomic DNA template. Lane 11: negative control, no PCR template.
