I used a spin column to extract DNA and as far as I know followed the protocol correctly. The Nanodrop has high peaks at 250 and 280 and a moderate peak at 260 nm. The 280 reading I am attributing to protein contamination but I did use proteinase K (20ul) in 180ul of sample and incubated at 57 degrees C for 4 1/2 hours. I did a enzyme digest with the sample and got no bands, no smear, nothing at all with staining in EtBr. I had other samples on the gel which came out fine so I know it is not the gel or stain or UV. Does anyone know what might have gone wrong and whether I can salvage anything from these samples?
lab geek
-alabgeek-
The problem may be that there are still Enzyme inhibitors in your DNA...
-gebirgsziege-
QUOTE (alabgeek @ Apr 1 2008, 03:11 AM)
I used a spin column to extract DNA and as far as I know followed the protocol correctly. The Nanodrop has high peaks at 250 and 280 and a moderate peak at 260 nm. The 280 reading I am attributing to protein contamination but I did use proteinase K (20ul) in 180ul of sample and incubated at 57 degrees C for 4 1/2 hours. I did a enzyme digest with the sample and got no bands, no smear, nothing at all with staining in EtBr. I had other samples on the gel which came out fine so I know it is not the gel or stain or UV. Does anyone know what might have gone wrong and whether I can salvage anything from these samples?
lab geek
Perhaps the proteinase K is the RE-inhibitor and/or the protein contamination? Sometimes people forget to remove, or not cleaning was not sufficent...
-hobglobin-
QUOTE (gebirgsziege @ Apr 3 2008, 07:51 AM)
The problem may be that there are still Enzyme inhibitors in your DNA...
What kind of inhibitors might they be and do you know what I could do to solve this problem?
-alabgeek-
QUOTE (hobglobin @ Apr 3 2008, 08:12 AM)
QUOTE (alabgeek @ Apr 1 2008, 03:11 AM)
I used a spin column to extract DNA and as far as I know followed the protocol correctly. The Nanodrop has high peaks at 250 and 280 and a moderate peak at 260 nm. The 280 reading I am attributing to protein contamination but I did use proteinase K (20ul) in 180ul of sample and incubated at 57 degrees C for 4 1/2 hours. I did a enzyme digest with the sample and got no bands, no smear, nothing at all with staining in EtBr. I had other samples on the gel which came out fine so I know it is not the gel or stain or UV. Does anyone know what might have gone wrong and whether I can salvage anything from these samples?
lab geek
Perhaps the proteinase K is the RE-inhibitor and/or the protein contamination? Sometimes people forget to remove, or not cleaning was not sufficent...
If the proteinase K is the problem...do you know if there is anything I can do to solve this?
-alabgeek-
to remove protK: phenol and/ or chloroform extraction.
-gebirgsziege-
QUOTE (alabgeek @ Apr 1 2008, 11:11 AM)
I used a spin column to extract DNA and as far as I know followed the protocol correctly. The Nanodrop has high peaks at 250 and 280 and a moderate peak at 260 nm. The 280 reading I am attributing to protein contamination but I did use proteinase K (20ul) in 180ul of sample and incubated at 57 degrees C for 4 1/2 hours. I did a enzyme digest with the sample and got no bands, no smear, nothing at all with staining in EtBr. I had other samples on the gel which came out fine so I know it is not the gel or stain or UV. Does anyone know what might have gone wrong and whether I can salvage anything from these samples?
lab geek
Do you need totally clean, protein-free DNA for the experiments? In most cases the DNA from spin columns is fine.
-swanny-
