I am not sure whether DTT will really improve the work of the proteinase K. Have you ever tried to increase the incubation time before adding the EtOH or is time a factor for you?

Thanks for the answer,

I did extend time incubation at 56°C from 10 to 30 minutes already but with minor results. Should I go for longer incubation times? I do not have time problem but with other cell types I get optimal results with just 10 mintues incubation, like reported in the manual.

kind regards

No, because you already increased the time it won´t help anymore. You can add more proteinase K, on the other hand 8 µg DNA is a lot of material.

Thanks a lot again for your considerations.

My major concerns are regarding two major aspects:

1- 8ug is overrated due to probably not so good spectrophotometric quantification (compared to what I see in agarose gel)

2- After purification I need to perform a precipitation step to concentrate the DNA for subsequent steps. As result of the precipitation I always loose about 50% of the DNA, so in the end not so much is left for analysis.

I tried to do precipitation as follows

- Add 1/10 volume 3M Na-acetate (pH 5.4)

- Add 10ug of glycogen (to help precipitation step)

-Add 2.5 volume ethanol

-Centrifuge 15' at 13000g

-Wash pellet with 70% ethanol

-Centrifuge 10' at 13000g

-Air dry and resuspend in TE (low E)

Thanks a lot for advice,

Leonardo