Bone samples Flashcards
What is one way to prepare a bone sample?
freezer milling (cryogenic)
Many extraction processes begin with steps aimed at?
removing contamination
After decontamination, the hard tissues are incubated in extraction buffer and?
proteinase K
What are some decontamination processes for bone samples?
- physically removing the outer bone surface by immersing in a bleach solution
- exposure to UV radiation
Why are the bone samples grinded to a powder?
increases the surface area to the various chemicals in DNA extraction to get more DNA
What is widely used for extraction of DNA from bone samples?
phenol-chloroform
After cleaning the bones with abrasion what is the next step to clean them?
soak in detergent
In the ball milling extraction process of bone samples what is used to freeze the sample?
liquid nitrogen
What are the two type of DNA recovery is used with bone samples?
- mortar and pestle
- cryogenic
What are the benefits of the cyrogenic method over the mortar and pestle?
- preservation of DNA integrity
- efficient cell distribution
- reduced contamination
- consistency in particle size
What are the advantages of the mortar and pestle method over cyrogenic?
- cost effective
- simple and traditional
- suitable for small samples
Why would we want to decalcify bone samples?
- facilitate cell lysis
- prevent DNA degradation
- improve efficiency
- enhance purity
- prevent interference
What are the overall steps in recovering DNA from bone samples?
- sample prep - clean exterior and remove contaminants
- decalcification using EDTA to remove calcium ions
- distruption of bone tissue - cryogenic/mortor and pestle
- cell lysis
- protein digestion - proteinase K
- DNA extraction using phenol-chloroform
- DNA purification
- quantification using a spectrophotometer or fluorometer
- quality check - agarose gel or capillary electrophoresis
