Nucleic Acid Extraction Methods

purpose of nucleic acid extraction

release nucleic acid from the cell to use for procedures and molecular testing

nucleic acid extraction should be free of —

proteins, lipids, DNA/RNA depending on which we are testing for

organic method of nucleic acid extraction

phenol-chloroform

• cheap

• more dangerous

• used in research labs

inorganic method of nucleic acid extraction

high salt concentration and low pH

• safer

• not as good as PCl

solid phase isolation (affinity)

uses solid matrix of silica based products to bind and wash DNA

• column or magnetic beads

• more rapid and effective

• wash before elution

what is in a nucleic acid extraction kit?

• lysis buffer - lyse cells

• proteinase K - break down proteins

• washer buffer 1 and 2

• elution buffer

lysis buffer contains—

salt, detergent (SDS), buffer (EDTA), proteinase K

lysis step of nucleic acid extraction incubation

10 minutes at 55 degrees

DNA precipitation solution

100% EtOH

wash buffer 1 for nucleic acid extraction

56% EtOH and salts

wash buffer 2 for nucleic acid extraction

70% EtOH

elution buffer

Tris buffer and EDTA

• more + charged than silica to pull off DNA

• incubate at RT for 5-10 mins to increase yield

why do we need to used molecular grade equipment?

make sure equipment is RNAse free to prevent RNA degradation

what matters in nucleic acid measurement?

quality and quantity

methods of nucleic acid measurement

gel electrophoresis, fluorometer, spectrophotometer, microfluidics

spectrophotometer is used to measure—

quantity of genetic material and purity

nucleic acids absorb light at—

260 nm

proteins absorb light at—

280 nm

what ratio is used to determine purity of extraction?

260/280

good DNA sample 260/280 ratio

1.8

good RNA sample 260/280 ratio

2.0

one optical unit at 260 nm is—

50 ug/mL of DNA

40 ug/mL of RNA

fluorometer

measures fluorescence related to DNA concentration by binding dye to DNA and fluorescing

DNA storage at RT

up to 4 months

DNA storage in refrigerator

1-3 years

DNA storage at -20 C

at most 7 years

DNA storage at -70 C

more than 7 years

RNA storage at RT or refrigerated

not recommended

RNA storage at -20 C

up to a month

RNA storage at -70 C

up to 30 days