Extraction

Question 1

What is extraction?

Answer 1

The lysing of cells to separate DNA from cellular material, followed by the isolation and purification of the DNA(Lyse->bind->wash->elute

Question 2

What are the reagents used for extraction?

Answer 2

ATL, PK, DTT, cRNA

Question 2

What is PK?

Answer 2

Proteinase K; an enzyme used to digest proteins(histones) to expose the DNA

Question 2

What is buffer ATL?

Answer 2

Tissue lysis buffer; used for the lysis of the cells to release and expose the DNA

Question 2

What is DTT?

Answer 2

Dithiothreitol; reduces the disulfide bonds of proteins allowing the release of DNA

Question 3

What are the digestion requirements of a trace, large volume, T->LV, and reference protocol?

Answer 3

10 minute digestion, 900rpm, 70 C

Question 4

What is the purpose of the EZ2?

Answer 4

This is used after the digestion to isolate and purify the DNA from the cellular material. Automated purification

Question 4

What are the digestion requirements of a hair protocol?

Answer 4

1 hour digestion, 900rpm, 56 C

Question 5

Explain the EZ2 process and what it does

Answer 5

DNA is isolated from lysates by binding to silica surface of magnetic particles in the presence of a Chaotrophic salt. DNA is then washed and eluted in an elution buffer

Question 6

What is cRNA?

Answer 6

carrier RNA;it enhances the binding of DNA to surface of magnetic particles, especially for low amounts of DNA

Question 7

How much sample is required for the different types of extractions?

Answer 7

Trace, hair, and reference (200uL); trace->LV 250uL from each sample which would add up to 500uL; Large volume 500ul