LAB - Nucleic Acid Extraction Flashcards
basic steps of DNA extraction/purification
- cel disruption/lysis
- purification of the nucleic acids from cellular material
- concentration of DNA
these disrupt non-covalent bonds in the proteins, denaturing them, and causing molecules to lose their native shape
detergents
chaotropic agent
molecule in water solution that can disrupt the hydrogen bonding between water molecules
disrupt the intramolecular bonds that fold proteins together = denaturant; effective at disrupting RNAse
- guanidine, chloride, phenol, ethanol
T or F. enzymatic digestion is a harsh lysis method
F! gentle
proteinase K
- enzymatic digestion; most common
- serine protease
- cleaves peptide bonds adjacent to the carboxylic group of aliphatic and aromatic AAs
lysozymes
- enzyme digestion; lyses gram +/- bacteria
- hydrolyzes peptidoglycan in bacterial cell wall
>B-1,4 linkages between NAM and NAG
Zymolase
enzyme digestion for yeast and fungal cell walls
Lysostaphin
lyse cell walls of Staphylococci
Lysostaphin
lyse cell walls of Staphylococci
Mutanolysin
lyse cell walls of Listeria and other gram + bacteria
RNAse A
- endonuclease
- cleaves phosphodiester bonds between 5’ ribose of a nucleotide and the phosphate group attached to the 3’ ribose of an adjacent pyrimidine nucleotide (cytosine and uracil)
- added at start or end after extraction
DNAse
- endonuclease
- cleaves DNA by preferentially acting on phosphodiester bonds or adjacent to pyrimidines to produce polynucleotides with terminal 5’-phosphates
nucleic acids can be precipitated out of solution by: (2)
- ethanol = routine; DNA more soluble in ethanol than isopropanol
- isopropanol = preferred for large vol extractions, less volatile than ethanol, pellets don’t stick
carrier molecules sor co-precipitants
- helps with precipitating really small nucleic acid molecules
- inert substances; using alcohol and salts
- can also enhance visualization of NA pellet after precipitation
- tRNA, glycogen, linear polyacrylamide, homopolymers of nucleotide poly dA
isolation of mitochondrial DNA
- enrichment = Mt DNa co-isolated with genomic DNA and is then preferentially amplified for enrichment
> efficient
> may introduce artifacts - differential centrifugation
> CsCl gradient and bis-benzamide fluorescent DNA dye
> time consuming
these must be lysed to extract DNA
cell wall and cellular membranes
Most commercial kits use a combination of ___, ___, and ___ ___ to lyse cells
- enzyme
- detergents
- chaotropic agents
nucleic acid solutions can be concentrated by “__ ___”
salting out; in the presence of alcohols
mRNA can be isolated by _____ chromatography
affinity
> polyA tail of eukaryotic messenger RNA binds to polyT or polyU chain
T or F. EDTA whole blood contains red and white blood cells, only the white bloods cells contain genomic DNA
T
Qiagen protease is a broad-specificity ______ protease with high activity, cleaving preferentially at ____ and _____ residues.The protease digest proteins and enzymes in the sample.
Buffer AL contains a SDS. SDS is a detergent and chaotropic agent it solubilized….
serine
neutral and acidic
lipids and denatures and solubilizes proteins
Buffer AL also contains ______ ______, another chaotropic salt which denatures proteins. This is also required for nucleic acid to adsorb to the silica membrane in the spin column.
guanidine hydrochloride
Buffers AW1 and AW2 contain ________.These wash buffers remove ….
ethanol
weakly bound reagents and cellular components from the membrane
The DNA can be eluted off the membrane using a … (Tris Cl EDTA pH 8) or molecular grade water.Tris Cl EDTA is better for long term storage as it prevents __ ________ to the DNA and the EDTA binds divalent cations which are required for _____ __________.
low salt buffer
pH damage
DNase activity
