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
what method does the QIAamp DNA isolation kit use to purify DNA?
salt/ precipitation method with binding membrane
what characteristic is notable when using the QIAamp DNA isolation kit compared to PCI methods?
lower molecular weight
what is the most common DNA isolation method used clinically?
kit based solid phase or magnetic bead isolations
what can cause low 260/280 ratio when measuring nucleic acids?
presence of phenolic solutions
what can cause high 260/280 ratio when measuring nucleic acids?
proteins
what method can be utilized to estimate the amount of DNA loaded on an agarose gel?
densitometry
how does densitometry estimate the amount of DNA in a band on an agarose gel?
based on the intensity of the band compared to the known standard
densitometry only works if what condition regarding the DNA bands on an agarose gel is met?
the bands are not saturated (overexposed)
what is the primary advantage of using gel analysis in DNA assessment?
it assesses DNA integrity
