LAB FUNDAMENTALS Flashcards
which enzymes are commonly used to break down proteins during the lysis step of ORGANIC DNA isolation
Proteinase K, protease, lysozyme
which chemicals are commonly used in organic DNA isolation methods?
phenol and chloroform; basically uses organic chemicals
what is the role of detergents in the lysis process of organic DNA isolation?
to disturb cell membranes
what are some examples of detergents used in organic DNA isolation?
SDS and Triton X-100
which component of PCI helps in defining the organic and aqueous phases in DNA isolation?
chloroform
from which layer is DNA typically extracted after centrifugation in the PCI phase of organic DNA isolation?
top layer (aqueous)
what type of salt is used in the organic isolation during precipitation?
sodium acetate and ammonium acetate
what role does salt play in ethanol precipitation of DNA in organic DNA isolation?
it promotes precipitation turning solid in solution
How is the DNA separated after centrifugation in the ethanol precipitation step or organic DNA isolation?
by washing in 70% ethanol
what does washing the DNA with 70% ethanol do?
hydrolyze the salt, but keep the DNA insoluble
what is the key principle behind solid phase DNA isolation?
DNA binds to a solid matrix or bead under high salt conditions
what is the most common manual method for solid phase DNA isolation?
spin columns
after lysing the cells and adding them to the column, what happens to the DNA?
the DNA binds to the column and the waste flows through.
what is the purpose of washing and spinning the column during the solid phase DNA isolation?
to remove contaminants from the column
what is the typical yield of highly purified DNA using the Qiagen QIAamp DNA isolation kit?
nanogram yield
