Download - DNA EXTRACTION from ANIMAL TISSUES
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