The procedure involves lysis of nuclear membrane and bind DNA on to the column. But DNA is tightly packed with histones as chromosomes are we disassociating DNA from histones? or extracting mutilated chromosome?
Hi Xin. Usually if you use a silica column-based kit the lysate is digested with Proteinase K in the presence of EDTA to inhibit DNases. A detergent is usually added during lysis to aid in denaturation of proteins and in solubilizing membrane proteins. Any residual RNA is removed by digestion with RNase prior to binding samples to the spin column.
Proteinase K is responsible for degrading proteins and separing DNA from them. Then the proteins are washed away and only the DNA stays "sticked" to the silica column.
An alternative to proteinase K may be multiple buffers that just separate proteins like histones from DNA. The proteins are then washed away.
So yes, histones are removed from DNA extraction and you should be able to extract whole genomic RNA (if you lose some "pieces" consider that you are extracting more cells, so you can "compensate").
Hi Xin. Usually if you use a silica column-based kit the lysate is digested with Proteinase K in the presence of EDTA to inhibit DNases. A detergent is usually added during lysis to aid in denaturation of proteins and in solubilizing membrane proteins. Any residual RNA is removed by digestion with RNase prior to binding samples to the spin column.
Proteinase K is responsible for degrading proteins and separing DNA from them. Then the proteins are washed away and only the DNA stays "sticked" to the silica column.
An alternative to proteinase K may be multiple buffers that just separate proteins like histones from DNA. The proteins are then washed away.
So yes, histones are removed from DNA extraction and you should be able to extract whole genomic RNA (if you lose some "pieces" consider that you are extracting more cells, so you can "compensate").
Read the product manual for your kit. FYI, there are LOTS of kit-based DNA extraction kits, not just Qiagen. You need to ask specific questions if you want specific answers.
Product manual doesn't mention anything about histones or chromosomes if it does you can ref. The kit manual just explains the way Umberto Palatini explained so I was asking in general. I'm aware they are several ways of DNA extracting. In short, I need to know whether DNA is free from histones. The question was very specific until some ppl started twisting it cuz they don't understand. Umberto Palatini answered the question with out complaining.
There is no page 50 in the document did you check before writing the comment. The document has 44 pages only. Maybe you could just quote that sentence or paragraph if you have difficulty with page numbers. The question was straight free from histones or not what is there to guess?
Purity and yield estimates follow after DNA isolation (from histones). How do we free DNA which is in heterochromatin/euchromatin form is the question not about yield or purity or different ways ("LOTS of kits") of extraction methods available and where exactly is that point mentioned anywhere in the manual it was not mentioned. Proteinase K frees DNA from the histones.
It was answered "So yes, histones are removed from DNA extraction and you should be able to extract whole genomic RNA (if you lose some "pieces" consider that you are extracting more cells, so you can "compensate")."
I managed to find the answer on RG https://www.researchgate.net/post/What_is_the_actual_role_of_Proteinase_K_in_human_DNA_extraction
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