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This is a modification of previous DNA protocols. It makes use of a high salt digestion buffer and samples are heated over night at high temperature to get the DNA to go into solution. Resulting DNA has been successfully used for Southern Blot analysis after Restriction Enzyme digestion.
Digest BufferFinal Conc. Stock for 500ml
100mM NaCl 5M 10ml
10mM Tris-Cl pH8 1M 5ml
25mM EDTA pH8 0.5M 25ml
0.5% SDS 20% 12.5ml
H2O 448ml
Protocol:
- Dilute Proteinase K (20mg/ml) 1:200 in the Digest Buffer to a final concentration of 0.1mg/ml.
- Once cells are confluent, rinse media off of cells, add 0.25ml of Digest Buffer containing PK.
- Incubate cells in the Digest Buffer/PK overnight @ 37˚C.
- At this stage, samples may be stored at -20˚C (this may actually help to get DNA into solution later).
- Add 0.5ml absolute EtOH to each well; swirl the plate vigorously - the DNA will form a spool at center of well (if the spool does not form it may be necessary to scrape the sample off the bottom of the well with a pipette tip).
- Using a fresh pipette tip for each sample, lift the spool of DNA out of the well and dunk it 4-5 times in 70% EtOH then 4-5 times in absolute EtOH; let the DNA dry only momentarily before placing the pellet into a microfuge tube containing 100µl TE (the volume of TE can be adjusted to the relative size of the DNA spools - this way a constant volume of DNA can be used in the RE digests and the DNA loads will be more consistent between samples without the need for quantitating every sample).
- Mix each sample by vortexing at max. speed for 5 min.
- Incubate all samples overnight in a waterbath @ 65˚C.
- Quick spin all samples and then check to see that each is fluid-like and that no DNA spool can be seen.
