DNA prep

DNA prep is done according to well established protocols.

• A 500 mg cell pellet is resuspended with 500 μl Cell Breaking Buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 100 mM Tris-HCl pH8.0, 1 mM EDTA) and distributed in 280μl aliquots.
• 200 μl Phenol:Chloroform:Isoamylalcool 25:25:1 and 300 μl glass 0.4-0.6mm unwashed glass beads are added to each aliquot.
• Samples are vortexed for 10 min at 4°C using a Disruptor Genie from Scientific Industrial (US Patent 5,707,861).
• 200 μl TE are added to each lysate, which are then centrifuged for 5 min at 16100x g, 4°C.
• The upper layer (~400 μl) is transferred to a fresh tube,
• 2.5vol 100% EtOH are added and the sample mixed by inversion.
• DNA is pelleted for 5 min at 16100x g, 20°C.
• The supernatant is removed and the pellets resuspended in 200 μl RNAse A 250 μg/ml for 15 min at 55°C, 1000 rpm on a Thermomixer comfort (Eppendorf).
• 2.5 vol 100% EtOH and 0.1vol NaOAc 3 M pH5.2 are added and the samples mixed by inversion.
• DNA is pelleted by centrifugation for 5 min at 16100x g, 20°C.
• The pellets are washed with 70% EtOH under the same conditions, the supernatant removed completely, and the pellets let dry for 10 min at 37°C.
• The pellets are resuspended in a total volume of 100 μl water for 10 min at 55°C, 700 rpm on a Thermomixer comfort (Eppendorf).
• DNA is run on a 0.6% 1X TBE agarose gel against a standard 1 kb GeneRuler, and quantified using FiJi. 500mg cell pellet should yield approximately 20-60 μg DNA. Always quantify on gel. Absorbance reading of this sort of "dirty" prep yields entirely meaningless results!!!

Agarose Gel of genomic DNA. Lanes 1-4 are 10, 8 4 and 2 μl of the marker (Generuler 1kb, 440ng/6μl). Lane 5 is 5μl of a 10x dilution of genomic DNA. Pictures should not be saturated when doing a gel quantification (unlike here...).