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DNA Isolation and Quantitation Cheat Sheet (DRAFT) by

This is a draft cheat sheet. It is a work in progress and is not finished yet.

Specimens in the Lab

Adequate for PCR
- Whole Blood
- Dried Blood
- Bone Marrow
- Saliva
- Tissue/ Scrapings
- Bone, Teeth
- Bacteria
- Amniotic fluid
- Fungi/­Yeasts
- Hair follicles/ Hair Shafts
- Buccal Cells
- Buccal Cells
- Hair
- Cerebr­ospinal fluid
- Urine
- Fixed tissue
- Stool
- Stool
- Soil
- Urine

DNA Extraction Methods

Uses organic chemical, phenol, cholor­oform
1. Lysis (NaOH, SDS)
2. Acidif­ication (acetic acid, salt)
3 Extraction (phenol, chloro­form)
4. DNA precip­itation (ethanol)
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
-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 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

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

DNA Quanti­tation

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!!
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.

Separating out the cells

Differ­etial Density gradient centri­fug­ation

Breaking open the cells

Alkaline lysis
Differ­ential lysis

Cell wall digestion