Posted 14 September 2009 - 04:51 PM
I use a 2% Incert agarose in TE, heated then cooled to 50C, a washed cell suspension heated to 37C, mixed in equal proportion (final agarose concentration 1%). I cut the end off a 1 ml syringe and load the agarose in the syringe, and let it cool. I slice 1-2 mm slices of the agarose with a clean razor blade and resuspend in TE. The number of cells in the initial cell suspension is important, and you should prepare several versions with different dilutions of cells. You want a slightly milky look, but not too white.
Then a series of digestions and washes are done, first with high EDTA concentrations with proteinase-K and N-laurylsarcosine, heated to 50C for 12 hours, then a set of washes with TE. Washes are also at 50C. Following washes, the cells can be stored at 4C for quite a while in TE. I did the washes in a hybridization oven rotator in 50 ml centrifuge tubes.
Digestion is done by washing at least 2x in RE buffer, followed by RE buffer + RE at 37C, followed by TE washes at 50C.
The gel slices can then be loaded into gel slots of a 1% gel, and sealed with hot 1% agarose.
You probably are overloading he gel, or the problem might be that the DNA is still circular. You can cut most genomic DNA at the ribosomal RNA sites with the enzyme I-CeuI, which is helpful to linearize fragment and identify the number of rRNA genes.
I'll try to dig up a real protocol for you.
Have you gotten the high MW yeast DNA fragments to run well on your setup?
What timing/voltage/angle are you using?