DNA EXTRACTION How does it actually work?
Dec 24, 2015
Cell Lysis
Lysis buffers are typically detergents such as sodium dodecyl sulfate (SDS)
What happens if you add dish soap to a frying pan with water that has a layer of grease on top?
Lysis Cont’d
Another analogy: popping a water balloon When you pop a
balloon, it is like lysing a cell
When the balloon is popped, all of the contents are released
Lysis Cont’d
So what actually happens?
Detergents disrupt lipid-lipid interactions compromising cell membrane integrity
Membrane is destroyed or becomes “leaky” depending on lysis buffer
Wash Stage
The next stage typically involves solutions to remove excess salts and impurities
Additionally, proteases, Rnases, etc. are often added to remove proteins (e.g., histones), RNA, etc.
Wash Cont’d
The wash solution (though separate solutions are sometimes used before) also adjusts pH
At low pH, DNA binds to a column Historically made of silica—a combination of
salts, silica and low pH create conditions that attract DNA
Elution
Elution is a fancy term for release of DNA from column
Water or TE (or other elution buffer) has a neutral or alkiline pH, leading to release of DNA