[[100kb+ ]] Genomic DNA preparation

Dave Hills david.hills at bbsrc.ac.uk
Wed Jun 17 04:43:18 EST 1998


If it's 100kb fragments you are after......then consider a different 
approach!! I would think that you will shear a lot of the DNA your way. 
Try the method that has been used for YAC/BAC library construction. This 
involves resuspending your cells in a low gelling temp agarose and then 
once this has solidified you can treat them whatever way you want. 
Typically to prepare DNA just place the agarose slabs in 0.5M EDTA with 
Proteinase K at 1mg/ml (expensive - but DNA is good!). You can then 
inhibit the proteinase and wash out the garbage with water/TE/EDTA etc. 
DNA is stable for months at least. Can treat DNA embedded in agarose with 
various enzymes to get your partial digested fragments of around 100kb. 
Gel purify those by PFGE.

I presume the hard part for you will be trying to produce a single cell 
suspension of the mosquito embryo. Can you break it up with trypsin etc?
Dave

Dr D Hills
Roslin Institute




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