L3: Nucleic Acid Extraction Methods

Nucleic Acid Extraction

To release nucleic acid from the cell for use in other procedures

Pure nucleic acid

what type of nucleic acids are used for nucleic acid extraction

Isolation

Routinely isolated from human, fungal, bacterial and viral sources

whole blood, tissue samples, micoorganisms

in isolation, Pretreat these specimens to make nucleated cells available

purified

in organic isolation, DNA Must be ? by removing contaminants

high salt; low pH; phenol and chloroform

Organic Isolation can be accomplished by using combination of ?, ? and an organic mixture of ?

RNAse

in organic isolation, To avoid RNA contamination add ?, enzyme that degrades RNA

Phenol/Chloroform

Biphasic emulsion forms (hydrophobic & hydrophilic)

Hydrophobic layer

what layer on bottom has cell debris

Hydrophilic

what layer on top has dissolved DNA

Salting out

Inorganic Isolation Methods Also called ?

Inorganic Isolation Methods

Uses low pH and high salt condition to selectively precipitate proteins

DNA

what is left in Inorganic Isolation Methods

isopropanol

in Inorganic Isolation Methods, dna is precipitated out using what solution?

snotty

description of DNA in salting out method

Solid Phase Isolation

isolation that is ► More rapid and effective ► Use solid matrix to bind the DNA. ► Wash away contaminants. ► Elute DNA from column

Crude lysis

what isolation technique is used for screening large no. of samples, isolate dna in small amounts and from challenging samples

Mitochondrial DNA

DNA that is passed from generation to generation along the maternal lineage

centrifuge, lyse, precipitate with cold ethanol

steps in Isolation of Mitochondrial DNA

labile

Isolation of RNA requires STRICT precautions to avoid sample degradation since RNA is ?

RNAses

Common laboratory contaminant: released from cellular compartments during isolation of RNA from biological samples ►Can be difficult to inactivate; small proteins that can renature and become active

Before

RNAses MUST be eliminated or inactivated (BEFORE or AFTER) isolation

wear gloves, use RNAse free tubes; ►Use dedicated, RNase-free, chemicals

Protecting Against RNAse

180 C; DEPC treated

for protection against RNAse, Pre-treat materials with extended heat for several hours, wash with ? water, NaOH or H2O2

ribosomal

►80-90% of total RNA is ? RNA

messenger

2.5-5% of total RNA iis ?RNA

1. Lyse/homogenize cells; 2. Add phenol:chloroform:isoamyl alcohol to lysed sample, and centrifuge; 3. Organic phase separates from aqueous phase 4. Remove RNA solution to a clean tube

steps in Organic RNA Extraction

polyA tail

Only 2.5-5%; mRNA molecules have a tail of A's at the 3' end

Oligo (dt)

these probes can be used to purify mRNA from other RNAs

Electrophoresis

Analyze DNA and RNA for quality based on their fluorescence

Ethidium bromide and SybreGreen

►Fluorescent dyes used in electropheresis

spectrophotometer

Sample absorbances are determined on the ? at 260nm and 280nm

260 nm

nucleic acid (DNA, RNA, nucleotides) absorb light at ?

280

protein absorbs light at ? nm

230

Guanidine absorbs light at ? nm

A260/A280

this ratio is a measure of DNA purity

directly

The absorbance wavelength is (directly or indirectly) proportional to the concentration of nucleic acid

260 Reading x Absorbance unit x Dilution factor

Formula Concentration

50 ug/mL

One optical density or absorbance unit at 260 nm is equal to ? in DNA

40 ug/mL

One optical density or absorbance unit at 260 nm is equal to ? in RNA

1.6-2.00

The OD at 260nm should be ? times more than the absorbance at 280nm

Fluorometry

utilizes fluorescent dyes which specifically bind DNA or RNA

negative control; standard of known conc.

Fluorometry requires what type of control and standard?

excitation

The fluorometer shines light on the sample, a process we called

Nucleic acids

The fluorescence of the dyes increases when they bind ?

1,000x

Fluorometry is about ? more sensitive than spectrophotometric absorbance

True

T/F: Do not use glass (spectrophotmetry) cuvettes in a fluorometer because the frosted glass