Cell Lysis buffer....do I have to add sodium phosphate to the buffer? - lysis buffer for DNA extractions (Sep/22/2008 )
I have been reading several protocols regarding cell lysis for DNA extraction from filters. Unfortunately I am bit confused because I am not sure what the right recipe for the lysis buffer is. It came down to two recipe, but I can't figure out which one to use:
1. final conc: 0.1M NaH2PO4, 0.1M Na2HPO4, 0.1M EDTA, 0.1M Tris-HCl, 1.5M NaCl
2. final conc: 0.1M Tris-HCl, 0.1M EDTA, 0.8M sucrose
Lysozyme, proteinase K and SDS are added at a later point for both protocols.
Is the sodium phosphate necessary for the lysis buffer? Any idea why sucrose?
I would be grateful for any input. Thanks!
Our lysis buffers donot contain sodium phosphate and one of our buffers is similar to the second recipe that you have with lower sucrose concentration. i think sucrose is to make the solution isotonic or hypertonic. Although we use the sucrose containing buffer when we need to extract some thing for some assays. If we plan some western, we donot add sucrose.
Thanks for your reply. I appreciate it! That was helpful.
If you look hard, the NaPo4 and NaCl just makes it PBS. I have used a cell lysis buffer that was PBS, with SDS, protease inhibitors etc.
The sucrose make a gradient to separate the DNA (top portion) from proteins and other debris.