I've been having issue with extracting/precipitating DNA from mouse tail pieces and am unsure if the lysis buffer I use could be the problem. After digesting for 2 hours with lysis buffer and Proteinase K I pour the supernatant into new tubes and add isopropyl alcohol for precipitation. Depending on how much liquid I pull out before drying the pellet and resuspending in water I see different levels of cloudiness as well as actual separation of contents in the tube. If I only take some liquid the solution is slightly cloudy, taking all of course leaves the tube clear after resuspension with water, and taking none results in a tube with two distinct layers with a white goop between the layers. Is this separation bad? Cloudiness never resulted when doing this previously even when leaving liquid behind which is why I think this step is the culprit.
Hello Peyton Hopkins
The lysis buffer should contain Tris HCl (pH 8.0), EDTA, NaCl and SDS. After the treatment with proteinase K and guanidine salt for protein denaturation you should centrifuge the tube and collect the entire supernatant. To the supernatant add ice-cold isopropyl alcohol and DNA will precipitate out. Centrifuge the tube and collect the DNA pellet. Dry the pellet so that the alcohol evaporates completely. Keep the tube at 37 deg in the incubator for complete evaporation of alcohol. Dissolve the DNA pellet in 1X TE buffer a pH 8.0.
Regards.
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