Proteinase K denaturation - (Oct/31/2007 )
can you tell what would happen if proteinase K was overheated to 90 - 100 °C during DNA isolation step. There was some proble with the water bath and it somehow overheated the samples. I anyhow went ahead with phenol-chloroform extraction and later upon isopropanol-Na-acetate precipitation, i could not see DNA molecule but the whole liquid turned somewhat milky. What can it possibly indicate (i.e. what could have possibly gone wrong). I think because proteinase K got denatured it didn´t perform the proteolysis but what i can´t understand is - the DNA is still there in the sample. why can´t it be seen?
I had a similar problem with bad Proteinase K, mine was from freeze-thawing a stock too many times. I also use phenol chl extraction to purify DNA. I actually thought I purified the DNA okay, but the quality was terrible and PCR failed. I think the since the Proteinase K was inactive, the DNA degraded. I'm not sure why your liquid was milky, maybe due to the heating at 95 and not the proteinase K degredation, but just wanted to let you know that the DNA quality probably isn't great anyway.
Hey actually it worked for me later on.
I had actually frozen the sample simply at - 20 for 1 and a half hours and then later centrifuged it and got a nice DNA pellet at the bottom.
Like i had predicted, the DNA was still there and somehow not visible. However, i performed ethanol wash step and will be measuring OD tomorrow to see the purity and then run a test gel. Hope it works.