large size genomic DNA isolation - (Sep/03/2007 )
i would like to isolate genomic DNA, but keeping it as much intact as possible. I dont want this DNA to be fragmented by the isolation procedure. Is there a protocol to keep it unfragmented as much as possible. I would like fragments larger than 1-2Mb.
please are there any suggestions?
To obtain DNA of this length, you must embed the cells in agarose and digest them in place. The resulting DNA is very fragile, and is difficult to manipulate in any way without breaking it. See any protocol for preparing pulse field gel electrophoresis (PFGE) samples.
thanks a lot phage434. thats what i have found too. protocols for PFGE.
however if you have a protocol that you can suggest to me i will be obliged.
The basic protocol is simple -- wash cells, resuspend in buffer at 37C. Prepare INCERT agarose at 2% and cool to 50C. Mix 50-50 and let the mixture cool in molds (I use a 3 ml syringe and then cut 1 mm slices). Lyse the cells with a sarkosine/EDTA/proteinase K mixture at 50C, followed by extensive washes with 500 mM EDTA at 50C. A hybridization oven works well for this. Store the slices in EDTA at 4C for long periods.
thanks a million. im already working on it!