DNA Isolation and Quantitation Cheat Sheet

Organic
Uses organic chemical, phenol, choloroform
1. Lysis (NaOH, SDS)	2. Acidification (acetic acid, salt)
3 Extraction (phenol, chloroform)	4. DNA precipitation (ethanol)
Inorganic
Uses inorganic chemicals, detergent, ethylenediamine tetraacetic acid (EDTA), acetic acid, salt (salting out, spooling)
1. Lysis (Tris, EDTA, SDS)	2. Protein precipitation (sodium acetate)
3. DNA precipitation (isopropanol)
Solid phase
DNA is immobilized on a solid support, beads, or columns
1. Lysis (supplied reagents)	2. Acidification (supplied reagents)
3. Adsorption (low pH)	4. Wash (supplied buffer)
5. Elute DNA (low salt)
Solid-Phase Isolation	-Kits -Spin Columns
Nucleic acid binding to silica beads in the basis for many automated extraction systems
Limiting specimens (fixed tissue, dried, bone)
Rapid extration for routine testing
Crude lysis
DNA does NOT need to be pure	RE digestion, gel electrophoresis, screening large amounts of DNA, some PCR, challenging specimens
Proteolytic enzymes	Proteinase K (PK)
Chelating resins	Chelex
Chelex resin
Heat tissue (hair roots, saliva, etc.) in 300 uL 5%-20% Chelex 100 resin
Chelex removes multivalent cations
DNA is in supernatant
Fixed tissue
1. ~20 micron sections	Macrodissect or microdissect
2. Digest (proteinase K, Tris buffer)

DNA isolation, purification and quantitation	Free of proteins, lipids, other nucleic acids
Different methods depending on specimen type	?????????????????????????????????????
Different methods depending on type of DNA needed	???????????????????????????????????????????

Spectrophotometry
Instrument that allows you to measure the amount of light that is absorbed in a sample or the amount of light transmitted based on the solute in the sample
Filters and/or prisms are used to filter the light to obtain a certain wavelength
A= absorbance %T= transmittance
A260/A280 Ratio
1.65 <= A260/A280 <= 2.50
dsDNA concentration = 50 ug/mL x OD260 x dilution factor
Beer - Lambert Law
Absorbance is directly proportionate to the concentration of the solute (DNA)
Absorptivity constant - 50 for DNA
- Conversion factor from optical density unit (absorbance units) to concentration
- Residual phenol - bad!!
Calculations
Usually have to dilute samples from preps to begin
A DNA prep diluted 1:100 has an A260 reading of 0.200. Find the concentration in ug/mL
A DNA prep diluted 1:50 has an A260 reading of 0.307. Find the concentration in ug/mL.

Differetial Density gradient centrifugation

Alkaline lysis
Differential lysis

DNA Isolation and Quantitation Cheat Sheet (DRAFT) by Morghay123