Escolar Documentos
Profissional Documentos
Cultura Documentos
DNA purification
column containing a
silica membrane
Silica
• DNA binds selectively to silica in the presence of high concentrations
of chaotropic salts (e.g., guanidinium HCl).
• Protein does not bind under these conditions.
• Silica membranes or columns are washed with an alcohol-based
solution to remove the salts.
• DNA is eluted from the membrane with a low-ionic-strength solution,
such as a low-salt buffer or water.
Advantages
• Fast purification
• Amenable to automation
• No centrifugation required (can use vacuum)
• No organic solvents or precipitation steps
Magnetic Separation: Silica or Charge-Based
Several commercial systems are
based on capture of DNA from
solution using magnetic particles.
Magnetic Particle Types
• Silica-based (bind/release DNA depending on
salt concentration)
• Charge-based (particle charge changes
based on pH of solution, binding/releasing
negatively charged DNA).
Advantages
• Fast Close-up view of the
silica-coated surface
• No organic extraction or precipitations
of a magnetic bead
• Amenable to automation
Anion-Exchange Columns
• Based on interaction between negatively charged phosphates in DNA
and positively charged particles.
• DNA binds under low-salt conditions.
• Protein and RNA are washed away using higher salt buffers.
• DNA is eluted with high salt (neutralizes negative charge on DNA).
• Eluted DNA is recovered by ethanol precipitation.
Advantages
• No organic solvents
• Fast, but more hands-on than silica (requires ethanol)
Example: Solution-Based Protocol
This example uses an easy, solution-based approach to
cell lysis, protein denaturation and DNA purification.
Centrifugation is used to remove precipitated materials
from solution.
View Animation
Example: Silica Membrane-Based Protocol
Here is an example protocol using a silica membrane to
capture DNA. Centrifugation or vacuum pressure is used to
pull materials through the membrane.
View Animation
DNA Quantitation
Once DNA is purified, it is usually quantified. Typical
quantities are in the milligram-picogram range
• gram (g)
• milligram (mg) = 10-3 g; 0.001g
• microgram (µg) = 10-3 mg; 0.000001g
• nanogram (ng) = 10-3 µg; 10-6 mg; 0.000000001g
• picogram (pg) = 10-3 ng; 10-6 µg; 10-9 mg; 0.000000000001g
Quantification Methods
Spectrophotometry: Use of light absorbance to measure
concentration. Many biological substances absorb light.
The spectrophometer measures absorbance of light at
specific wavelengths
• Most commonly used method
• DNA concentration can be calculated based on
absorbance at 260 nm:
A = e × c × l (Beer-Lambert Law)
A = absorbance
e = extinction coefficient
c = concentration
l = path length
DNA Quantitation
Common conversions
• Double-stranded DNA: 1 A260 = 50 µg/ml
• Single-stranded DNA: 1 A260 = 33 µg/ml
DNA Quantification
High-sensitivity fluorescence methods are also
available
• PicoGreen® method. Mix sample with reagent, wait
5 minutes and read with a fluorimeter.