Extraction and Purification of nucleic acids-RNA

Question 1

What are the 5 steps of RNA Extraction

Answer 1

-Sample collection
-Cell Lysis
-Phase separation
-RNA Precipitation
-RNA resuspension

Question 2

What is the RNA Stabilizing agents temperature that is commonly used

Answer 2

(-80 C) Invitrogen

Question 3

The cells or tissues are lysed using…

Answer 3

Detergents and Chaotropic agents

Question 4

What are the 3 mechanical disruption methods

Answer 4

Sonication
Bead beating
Homogenization

Question 5

The most common method involves________ using reagents____

Answer 5

-Phenol chloroform extraction
-Reagents: TRIzol

Question 6

What are the 3 phenol chloroform that creates biphasic separation:

Answer 6

Aqueous phase
Interphase
Organic Phase

Question 7

RNA is precipitated by_____ in the presence of salts like sodium acetate or lithium chloride.

Answer 7

Isopropanol or ethanol

Question 8

The_____ is obtained by centrifugation and watch with _____to remove salts

Answer 8

RNA pellet and 70% ethanol

Question 9

Three commonly used methods can effectively Isolate RNA from samples

Answer 9

-The guanidinium thiocyanate-based method
-Spin Column based method
-The combined guanidinium thiocyanate-column based method

Question 10

Chaotropic agent used in protein
degradation

Answer 10

Guanidinium thiocyanate

Question 11

RNA is separated from DNA after extraction with acidic solution consisting

Answer 11

Guanidinium thiocyanate and phenol

Question 12

These are much faster, avoid the use of the toxic phenol and chloroform reagents.

Answer 12

Spin column method

Question 13

It is the template for protein translation, and most of the eukaryotic mRNAscarry tracts of it at their 30 termini.

Answer 13

Isolation of poly A+ RNA

Question 13

Two methods are commonly used in the selection of poly(A)+ RNA—

Answer 13

Column Chromatography and batch chromatography

Question 13

is normally used for the
purification of large
quantities (>25μg) of
poly(A)+ RNA isolated
from mammaliancells.

Answer 13

Column Chromatography

Question 13

is the preferred method when
working with small amounts (<50μg) of total mammalian
RNA

Answer 13

Batch Chromatography

Question 14

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)

Answer 14

Magnetic oligo dt bead

Question 14

Enzymatic RNA removal that be used:

Answer 14

RNase A
RNase H
RNase I
RNase T1

Question 14

separates proteins and nucleic acids

Answer 14

Isoamyl alcohol

Question 14

*A260/A280 ratio (Protein contamination check) →

Answer 14

1.2-2.0

Question 14

*A260/A230 ratio (Salt and phenol contamination check) → Optimal:

Answer 14

> 2.0

Question 15

Degrades single-stranded RNA but not DNA

Answer 15

RNase A

Question 16

Specifically degrades RNA in RNA-DNA hybrids

Answer 16

RNase H

Question 17

Cleave RNA at specific
nucleotide sequences

Answer 17

RNase I or RNase T1

Question 18

effectively lyses cells and inactivates RNases, preventing RNA degradation.

Answer 18

Guanidinium thiocyanate

Question 26

Selectively binds to RNA to silica membranes, removing contaminants like DNA, proteins, and lipids

Answer 26

Spin column based purification