Role of Proteinase K during DNA isolation - (Mar/10/2005 )
In our lab we are studying cell-death related pathways. Before starting key experiments, first we are trying to detect DNA ladder formation after drug treatment. By the conventional DNA isolation method, we don't see any bands. Due to the temporary shortage of Proteinase K, we skipped the PK treatment. Do you think it might have caused the trouble? In my opinion, PK is very important for the isolation of relatively high molecular weight DNAs and is useful for removing the interaction between DNAs and histones. What is your opinion? What do you think about the role of PK during DNA isolation. Please let me have the truth.
* I performed the extraction process without RNase treatment.
-virusyoung-
QUOTE (virusyoung @ Mar 10 2005, 06:17 PM)
In our lab we are studying cell-death related pathways. Before starting key experiments, first we are trying to detect DNA ladder formation after drug treatment. By the conventional DNA isolation method, we don't see any bands. Due to the temporary shortage of Proteinase K, we skipped the PK treatment. Do you think it might have caused the trouble? In my opinion, PK is very important for the isolation of relatively high molecular weight DNAs and is useful for removing the interaction between DNAs and histones. What is your opinion? What do you think about the role of PK during DNA isolation. Please let me have the truth.
* I performed the extraction process without RNase treatment.
* I performed the extraction process without RNase treatment.
Proteinase K is used in DNA extraction to digest the tissue/cell membranes, etc. If the tissue and/or cells aren't digested all the way, it will greatly decrease your yield (it sounds like yours weren't digested at all which is probably why you didn't get any bands). It could very well digest histone proteins as well, but I'm not sure about them specifically. Hope this helps.
-ladybug-