DNA EXTRACTION from ANIMAL TISSUES Sahara Nesli Gonzaga

Bernadette Diane VistaGroup 5, WAD1

Introduction:• DNA, or deoxyribonucleic acid, is

the hereditary material in humans and almost all other organisms.

the continuing storage of information: comprises instructions needed for the synthesis of other cell organelles and other constituents.

Introduction:

composed of two long polymers of simple units, the nucleotides, with sugar and phosphate groups linked together by ester bonds as the backbone

Introduction:• Extraction of DNA: removal of DNA

from cells or viruses from which it normally inhabits.

• Norman Wang Method: one step extraction of DNA from agarose gel electrophoresis without Gel Cutting & Purification.

Introduction:• UVSpectrophotometry:

quantitative measurement of the reflection or transmission properties of a material as a function of wavelength.

• Agarose Gel Electrophoresis: separate DNA fragments based on its molecular weight, charge and shape.

Methodology: Extraction of DNA 8mL Blood + EDTA-Na

0.5mL aliquots + 0.5mL Wang’s lysis solution

Centrifuge (10,000 x g; 20s)

Pellet + 1mL lysis solution

Mix & centrifuge (10,000 x g; 20s)

Pellet and Supernatant (repeat procedure)

Methodology: Extraction of DNA Pellet + 0.2mL enzyme

solution Incubated (37o ; 10min)

+ 10μl Proteinase K solution ; incubated

+0.3ml sodium iodine (NaI) solution

+ 0.5ml isopropanol Mixed by inversion

Methodology: Extraction of DNA Centrifuge (10,000 x g;

20s)remaining alcohol was

aspirated

Pellet + 1ml of isopropanol

Centrifuge (10,000 x g; 5min)

Repeat procedure: 70% ethanol

Pellet + 0.1ml TE (Tris-EDTA)

Methodology:

UV spectrophotometer

http://www.spectralabsci.com/images/PE-UV-Vis-Spectrophotometers-Lambda-12.jpg

5uL of DNA sample diluted in 495 uL of TE

buffer

Recording of absorption readings

Methodology:

Agarose Gel Electrophoresis

http://upload.wikimedia.org/wikipedia/commons/a/a6/Gel_electrophoresis_apparatus.JPG

Powdered agarose: 1x TAE

comb was placed (30-40min)

+ EtBr (0.5ug/ml)

Methodology:

Agarose Gel Electrophoresis

http://upload.wikimedia.org/wikipedia/commons/a/a6/Gel_electrophoresis_apparatus.JPG

+1x TAE buffer (depth of at least 1mm)

sample DNA mixed with the desired gel loading

buffer

viewed under UV light with the use of a UV transilluminator.

Results & Discussion

Results & Discussion

Results & Discussion

Results & Discussion

Conclusion:• DNA is an important type of nucleic

acid essential to any living organisms metabolism, function and development.

• There are numerous ways to extract DNA depending upon the sample from which it will be taken.

Conclusion:• UV spectrophometer basically aids

in analyzing DNA extract through its absorbance (ability to absorb light) in a given wavelength

• Agarose Gel Electrophoresis separates molecules through their difference in charge, size and shape.

Guide Questions:

Guide Questions:2. What is the principle behind agarose gel

electrophoresis?

separates molecules based upon charge, size and shape

A direct current power supply is connected to the electrophoresis apparatus and current is applied.

The higher the applied voltage, the faster the samples migrate.

The buffer serves as a conductor of electricity and to control pH.

Guide Questions:2. What is the principle behind agarose gel

electrophoresis?

Agarose is a polysaccharide derivative of agar: mixture of agarsoe and hydrocoloids.

Molecules having a more compact shape can move more easily through the pores.

Charge, size, shape together with buffer conditions, gel concentration and voltage, affects the mobility of molecules in gels.

Guide Questions:3. Suggest other ways to purify animal DNA aside from

the above-mentioned procedure and commercially available kits.

Purification by silica: DNA of interest can be isolated by virtue of its ability to bind silica in the presence of high concentrations of chaotropic salts.

Plasmid DNA purification: the difference in denaturation and renaturation characteristics of covalently closed circular plasmid DNA and chromosomal DNA fragments.

Guide Questions:3. Suggest other ways to purify animal DNA aside from

the above-mentioned procedure and commercially available kits.

Genomic DNA isolation: solution-based Wizard® Genomic DNA Purification Kit, relies on a series of precipitation steps to purify high-molecular-weight DNA from a prepared lysate.

References:• Carpi, A., & Ph.D.. (n.d.). Nucleic Acids. Visionlearning.

Retrieved February 15, 2011, from http://www.visionlearning.com/library/module_viewer.php?mid=63

• DNA Purification Protocols and Applications Guide. (n.d.). Promega Corporation - Precision

• DNA structure. (n.d.). Wickipedia. Retrieved February 15, 2011, from commons.wikimedia.org/wiki/File:DNA_Structure.jpg http://ghr.nlm.nih.gov/handbook/basics/dna

References:• Principle and practice of agarose gel electrophoresis.

(n.d.). gannon. Retrieved February 15, 2011, from www.gannon.edu/resource/dept/sim/new/biologyexp_files/bio_exp_pdf/electrophoresis%20-%20principles%20and%20practi

• Design for Life. Retrieved February 15, 2011, from http://www.promega.com/paguide/chap9.htm

• Principles of Gel Electrophoresis. (n.d.). arbl.cvmbs.colostate.edu. Retrieved February 15, 2011, from http://www.vivo.colostate.edu/hbooks/genetics/biotech/gels/principles.html