Freezer milling Flashcards
What is freezer milling?
- Freezer milling is a technique used for processing biological samples, including bone samples, under extremely cold conditions.
- In freezer milling, the sample is frozen to very low temperatures using liquid nitrogen or other cryogenic agents before being mechanically milled or pulverized.
Freezer milling steps
- Freezing the sample
- Mechanical milling
- Sample collection
Step 1
Freezer milling
Freezing the sample
The bone sample is placed in a container or vessel and immersed in liquid nitrogen or another cryogenic agent. This rapidly lowers the temperature of the sample to well below freezing, often to temperatures approaching -196°C.
Step 2
Freezer milling
Mechanical milling
Once the sample is frozen, it is mechanically milled using specialized equipment such as a cryogenic mill or grinder. This equipment is designed to operate at low temperatures and can effectively grind or pulverize the frozen sample into fine particles or powder.
Step 3
Freezer milling
Sample collection
The milled sample is collected for further analysis or processing. The fine particles or powder obtained through cryomilling can be used for various downstream applications, such as DNA or RNA extraction, protein analysis, histological studies, or elemental analysis.
Advantages of freezer milling for processing bone samples
- Preservation of Sample Integrity: Freezing the sample helps preserve its molecular and structural integrity by minimizing degradation and enzymatic activity.
- Efficient Grinding: Cryogenic milling allows for efficient grinding of hard or difficult-to-process materials, such as bone, by making them more brittle and easier to pulverize.
- Reduction of Contamination: The low temperatures used in cryomilling can help reduce the risk of contamination and microbial growth in the sample.
When is freezer milling useful?
In forensics and disaster victim identification
Immediately after the cell dies, DNA will begin to?
Degrade
Hard tissue, which stabilises DNA provides protection against?
Degradation
Many extraction processes begin with steps aimed at?
Removing contamination
After decontamination, the hard tissues are subsequently incubated in extraction buffer and?
Proteinase K
Non-coding, repeating DNA sequences?
STRs
Freezermill 6870D
Sample processes
- Place sample in glass vial and add stainless steel cap
- Dual grinding of sample
- Dual pre cooling chambers to cryogenic temepratures
- The sample is pulvisrised by the magnetically shuttled steel impactor that does back and fourth against steel end plugs
- Liquid nitrogen keeps the sample cold preserving its integrity.
- Once pulverized its ready to be analysed.
Most common nucleated cells in bone matrix?
Osteocytes
Widely used for extraction of DNA from bone sample?
Phenol-chloroform
Remove contaminants by cleaning with?
Bleach
Cleaning the bones with abrasion can be followed by soaking in?
Abrasion and detergent
Next step after extraction
Quantification
Decontamination process for bone samples
Physically removing the outer bone surface by immersing in a bleach solution or exposure to UV radiation.
Why are bone samples grinded to a powder?
increases the surface area to the various chemicals in DNA extraction to get more DNA
What does hard tissue provide for DNA?
The hard tissue of the body, such as teeth and bones, provide a refuge for DNA
Advantage of hard tissue over other forms of biological material?
- Hard tissues provide an advantage over biological material because they have a hard surface that can be cleaned to remove contaminating DNA by using detergent to remove any soft tissue.
After DNA contaminants have been removed off hard tissue, what is the next step?
Physical abrasion using bleach and exposure to strong UV light for further cleaning.
Once cleaning of the bones is done, what is next?
The material is broken down into a powder via drilling or grinding under liquid nitrogen.
