DNA extraction using qiagen kit - (May/23/2013 )
Hallo all,
I am doing a DNA extraction with a qiagen kit (http://www.qiagen.com/Products/Catalog/Sample-Technologies/DNA-Sample-Technologies/Genomic-DNA/DNeasy-Blood-and-Tissue-Kit#orderinginformation).
Now I wonder what the ATL and AL buffers are.
Also the AW1 and AW2 buffer.
What I found so far is:
ATL : animal tissue lysis. This is pretty straight forward and I understand this.
AL: here is where I get confused => I found (googling) its a cell lysis buffer ? But why would I need another lysis buffer? Isnt ATL used to lyse the tissue and cells? or?
AW1: used to denature the proteins so they can pass through the filter
AW2: 70% ethanol to wash the salts out. It also contains a qiagen concentate.. but no idea what that means..
Any ideas about that AL buffer ?
First of all I don't think it's really necessary to composition of QIAGEN buffers, as long as it works for you. Many of them are proprietary anyway.
From what I found AL buffer containing guanidine salts for better column binding and detergent.
AW1 and 2 are wash buffers supplied as concentrates, AW1 contains is a stringent wash with low concentration of quanidine and AW2 is a Tris-based etanol solution to remove salts.
Trof on Thu May 23 19:26:24 2013 said:
First of all I don't think it's really necessary to composition of QIAGEN buffers, as long as it works for you. Many of them are proprietary anyway.
From what I found AL buffer containing guanidine salts for better column binding and detergent.
AW1 and 2 are wash buffers supplied as concentrates, AW1 contains is a stringent wash with low concentration of quanidine and AW2 is a Tris-based etanol solution to remove salts.
I can understand, but I do wonder what the difference is between tissue lysis buffer (ATL) and lysis buffer (AL).
Seems pretty weird to use two lysis buffers.
Not really. If you'd be doing the isolation yourself, you would probably use specific lysis buffer for each case, but commercial kit can't waste buffers you won't use. So they made it with one all-purpose necessary lysis and equilibration buffer and one probably stronger only for tissues.
Commercial kit protocols are optimized for the way and width of their use, which may seem less strightforward from a view of a common lab practice.
FYI the Qiagen ATL buffer is mainly SDS, and it serves to destroy nuclease activity without hurting the Proteinase K (PK) too much, and helping PK digest proteins. You could add 5-10mM CaCl2 to help the PK and maybe trace EDTA (~2mM) to pull away Mg2+ from nucleases but the 2-5% SDS plus PK really trashes the protein (and Hb in blood) while the nuclease die. RNAase A will similarly die. ATL is also pH 8.6 (likely Tris) and looks to be similar to "tail tip" digestion buffer. AL addition (~4M GuHCl) puts the digested protein in solution and then ETOH addition (to ~33%) helps keep the SDS in solution as it tends to precipitate with GuHCl.