Freezer milling

Question 1

What is freezer milling?

Answer 1

• 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.

Question 2

Freezer milling steps

Answer 2

1. Freezing the sample
2. Mechanical milling
3. Sample collection

Question 3

Step 1

Freezer milling

Freezing the sample

Answer 3

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.

Question 4

Step 2

Freezer milling

Mechanical milling

Answer 4

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.

Question 5

Step 3

Freezer milling

Sample collection

Answer 5

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.

Question 6

Advantages of freezer milling for processing bone samples

Answer 6

• 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.

Question 7

When is freezer milling useful?

Answer 7

In forensics and disaster victim identification

Question 8

Immediately after the cell dies, DNA will begin to?

Answer 8

Degrade

Question 9

Hard tissue, which stabilises DNA provides protection against?

Answer 9

Degradation

Question 10

Many extraction processes begin with steps aimed at?

Answer 10

Removing contamination

Question 11

After decontamination, the hard tissues are subsequently incubated in extraction buffer and?

Answer 11

Proteinase K

Question 12

Non-coding, repeating DNA sequences?

Answer 12

STRs

Question 13

Freezermill 6870D

Sample processes

Answer 13

• 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.

Question 14

Most common nucleated cells in bone matrix?

Answer 14

Osteocytes

Question 15

Widely used for extraction of DNA from bone sample?

Answer 15

Phenol-chloroform

Question 16

Remove contaminants by cleaning with?

Answer 16

Bleach

Question 17

Cleaning the bones with abrasion can be followed by soaking in?

Answer 17

Abrasion and detergent

Question 18

Next step after extraction

Answer 18

Quantification

Question 19

Decontamination process for bone samples

Answer 19

Physically removing the outer bone surface by immersing in a bleach solution or exposure to UV radiation.

Question 20

Why are bone samples grinded to a powder?

Answer 20

increases the surface area to the various chemicals in DNA extraction to get more DNA

Question 21

What does hard tissue provide for DNA?

Answer 21

The hard tissue of the body, such as teeth and bones, provide a refuge for DNA

Question 22

Advantage of hard tissue over other forms of biological material?

Answer 22

• 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.

Question 23

After DNA contaminants have been removed off hard tissue, what is the next step?

Answer 23

Physical abrasion using bleach and exposure to strong UV light for further cleaning.

Question 24

Once cleaning of the bones is done, what is next?

Answer 24

The material is broken down into a powder via drilling or grinding under liquid nitrogen.

Question 25

What happens to the bones once they’ve been drilled?

Answer 25

The resulting material is decalcified using EDTA either before or at the same time of cell lysis.

Question 27

What is a disadvantage of extracting DNA from bones?

Answer 27

It takes much longer than extracting DNA from bone than with other forms of samples.

Question 28

What does exposure to UV light do to bone samples?

Answer 28

It introduces thymine dimers into any contaminating exogenous DNA and stopping it from being amplified during PCR.