Extraction and Purification of nucleic acids-RNA Flashcards
What are the 5 steps of RNA Extraction
-Sample collection
-Cell Lysis
-Phase separation
-RNA Precipitation
-RNA resuspension
What is the RNA Stabilizing agents temperature that is commonly used
(-80 C) Invitrogen
The cells or tissues are lysed using…
Detergents and Chaotropic agents
What are the 3 mechanical disruption methods
Sonication
Bead beating
Homogenization
The most common method involves________ using reagents____
-Phenol chloroform extraction
-Reagents: TRIzol
What are the 3 phenol chloroform that creates biphasic separation:
Aqueous phase
Interphase
Organic Phase
RNA is precipitated by_____ in the presence of salts like sodium acetate or lithium chloride.
Isopropanol or ethanol
The_____ is obtained by centrifugation and watch with _____to remove salts
RNA pellet and 70% ethanol
Three commonly used methods can effectively Isolate RNA from samples
-The guanidinium thiocyanate-based method
-Spin Column based method
-The combined guanidinium thiocyanate-column based method
Chaotropic agent used in protein
degradation
Guanidinium thiocyanate
RNA is separated from DNA after extraction with acidic solution consisting
Guanidinium thiocyanate and phenol
These are much faster, avoid the use of the toxic phenol and chloroform reagents.
Spin column method
It is the template for protein translation, and most of the eukaryotic mRNAscarry tracts of it at their 30 termini.
Isolation of poly A+ RNA
Two methods are commonly used in the selection of poly(A)+ RNA—
Column Chromatography and batch chromatography
is normally used for the
purification of large
quantities (>25μg) of
poly(A)+ RNA isolated
from mammaliancells.
Column Chromatography
is the preferred method when
working with small amounts (<50μg) of total mammalian
RNA
Batch Chromatography
alternative for the purification of poly(A)+ RNA from total RNA sample. The poly(A)+ RNA can be
extracted by introducing magnetic beads coated with oligo(dT)
Magnetic oligo dt bead
Enzymatic RNA removal that be used:
RNase A
RNase H
RNase I
RNase T1
separates proteins and nucleic acids
Isoamyl alcohol
*A260/A280 ratio (Protein contamination check) →
1.2-2.0
*A260/A230 ratio (Salt and phenol contamination check) → Optimal:
> 2.0
Degrades single-stranded RNA but not DNA
RNase A
Specifically degrades RNA in RNA-DNA hybrids
RNase H
Cleave RNA at specific
nucleotide sequences
RNase I or RNase T1
effectively lyses cells and
inactivates RNases, preventing RNA degradation.
Guanidinium thiocyanate
Selectively binds to RNA to silica membranes, removing contaminants like DNA, proteins, and lipids
Spin column based purification
