Most commercial genomic DNA extraction kits used today seem designed for microscale genomic DNA extraction, e.g. for genotyping. I am looking to extract DNA from e.g. mouse spleen or any other appropriate tissue, for size selection of genomic restriction fragments to prepare a limited genomic library to determine transgene insertion sites and the nature of the flanking DNA. Thus I am looking for a convenient modern day kit equivalent of the old style medium scale genomic DNA extraction carried out previously by Proteinase K and RNase A digestion of homogenised tissue, followed by phenol extraction, ethanol precipitation and resolubilisation. Any suggestions based on recent experience would be greatly appreciated.
Various brand available such as Qiagen, Promega...
https://www.promega.com/resources/product-guides-and-selectors/protocols-and-applications-guide/dna-purification/
Thanks
Many thanks for your various responses and your tolerance of my rather outmoded desire to isolate microgram amounts of genomic DNA for genomic fragment size selection and Southern Blotting. I can only justify my embarrassment by explaining that I am seeking to clone and analyse a unique transgene integration site for which there are currently are no reagents, although I hope to be able to design a multiplex PCR assay for heterozygous and homozygous insertions once I know the sequence of the chromosomal regions flanking the transgene insertion site.
The Promega DNA purification Applications Guide is a great document to have access to, not least because of its historical review of the hazardous reagents that we used on a daily basis 'when I were a lad', btw 'young post-doc'!
The solution-based Promega Wizard™ Genomic DNA Purification Kit seems both flexible and scalable but lacks some key reagents, such as Proteinase K, that need to be provided by the user. However, the MasterPure™ Complete DNA and RNA Purification Kit is most obviously adaptable to scale and seems to include all the necessary reagents for DNA isolation from mouse tissues. In contrast, I could not locate anything in the Qiagen range catering above the microscale.
I'll probably be giving the MasterPure kit a crack, while feeling misty-eyed for the days of isolating milligram of DNA for cloning and library construction, but still keen to receive any further feedback that is out there.
Best wishes
Steve Morley
- Badges
- Science topic
- Similar topics
- Biological Science
- Bioecology
- Systems Ecology
- Landscape Ecology
- Connectivity