High Molecular Weight DNA extraction and PFGE - (May/08/2013 )
I'm currently using different techniques to extract high molecular weight DNA (up to around 120kb) from Streptomyces spp. and am finding the literature rather lacking in alternative methodologies.
Using a kit is unfotunately not an option.
Any tips and tricks would be greatly appreciated.
I'm trying to make a BAC library at the moment and I followed this protocol - chapter 7 (http://wheat.pw.usda.gov/jag/papers00/paper300/pdf/BACmanual.pdf) to extract HMW DNA. The procedure worked well and I obtained a high quality and concentrated DNA. Have a look at that!
Do you have any experience in pulsed field electrophoresis? I've been experiencing a lot of problems in separating the PFG marker. Even modifying switch times/voltage, agarose percentage on gel and running buffer it still does not work. Either the marker does not run straight or separetes just a little , even below a high voltage and long run time. Any tips about that?
What is your marker? Is the buffer cooled? The NEB yeast chromosome marker works very well in our hands. The lambda marker works less well.