Genomic DNA, isolation

Varela-Alvarez E, Andreakis N, Lago-Leston A, Pearson GA, Serrao EA (2006) Genomic DNA isolation from green and brown algae (Caulerpales and Fucales) for microsatellite library construction. J Phycol 42 741-745... 

Examining the Rate of Renaturation for Genomic DNA Isolated from E. Coli. 129... 

Because of the sequence complexity of genomic DNA, isolation of false positive clones by blot hybridization is a significant problem when the stringency of the washing conditions is very low (wash temperature <42°C). 

Testing can be performed on genomic DNA isolated from the entire ceU population of the specimen. Alternatively, the sample can be sorted by flow cytometry or immunorosetting to assess engraftment of specific ceil lineages. For example, engraftment of the T-cell versus non-T-cell components can be assessed after flow cytometry sorting based on expression of the CD3 surface molecule, a T-ceU marker. 

The system was demonstrated by performing an amplification of the p-globin gene from human genomic DNA isolated from whole blood. The RT-PCR products were analyzed off-chip using agarose gel (2%) electrophoresis and ethidium bromide staining. 

The major steps in the CGH protocol include genomic DNA isolation and labeling, microarray hybridization, scanning of slides and calculation of fluorescence intensity ratios, and data analysis by use of appropriate statistical methods (see Fig. 1). 

Perform genomic DNA isolation from S. aureus test and reference strains using a modification of the Edge Biosystems genomic DNA isolation kit. The modification consists of addition of lysostaphin (100 pg/mL final concentration) to bacterial cells and incubation at 37 °C for 10 min prior to DNA extraction. 

The genomic DNA isolated is a suitable substrate for digestion with a variety of restriction enzymes. Ideally a restriction enzyme is chosen which cuts frequently in most genomic DNA but not within tandemly repeated minisatellite sequences, a variety of such four base pair restriction enzymes exist, e.g. Hae III, Alu I and Hint I, although their suitability varies with different species. 

Transgenic animals are identified by Southern Blot or PCR analysis of genomic DNA isolate from the tail tissue. 

Perform multiplex PCRs following the manual for the Qiagen Multiplex PCR Kit. This often requires htde to no optimization. Use no template and yeast host DNA as negative controls. Use genomic DNA isolated from the bacterium of interest as a positive control. As another positive control, mix together yeast host DNA and genomic DNA isolated from the bacterium of interest. 

All genomic DNA isolations used in the generation of RAPD-PCR profiles by our research group are made with QIAquick PCR purfication kits (Qiagen). The resulting pellets are washed with 70% ethanol, dried and resuspended in 50 iL of TE [lOmM TRIS pH 8.3), 1 mM EDTA]. The quantities of DNA isolated for each sample are estimated by electrophoresis on a 1% agarose yield gel. Upon dilution to make DNA concentrations consistent between samples, all isolates are either immediately utilized or frozen at -20° for later use. 

Figure 1. RAPD-PCR profiles of crayfish collected from an impacted site and one of its associated reference sites. PCR products generated using the A-01 primer (Operon Technology) on genomic DNA isolated from six crayfish collected at a reference site (ISI value 36) on the Ottawa River (lanes 2-7), and from six crayfish collected at a downstream impacted site (IBI value 21) on the same river (lanes 9-15). Molecular size markers are included in lanes I, 8 and 16 and are indicated at left.

Fig. 2 Results of PCR reactions with genome DNA isolated from bone scar after MSC transplantation. M ladder, C positive control, 1, 2, 3, 4, 5, 6 and 7 7 mice grafted with MSC...

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