Question

Question Set:pDNA Isolation Data: Sample A: 0.02 Az00: 0.013 Dilution: To on 101 Concentration: 1. Lushal Questions: 1. What is RNase A and why was it added to the PDI Buffer? 2. Alcohol (EtOH) was added to the column Wash Buffer. Why? What does this do to the chromosomal DNA? 3. What structure collects the DNA by binding to the matrix? 4. Explain how detergents are used in the PD2 Buffer. 5. Name two methods for detecting DNA isolated in this week's lab.

Answer 1

Que : what is RNase A and why it added to PD1 buffer?

• RNase is the ribonulease. It is an catalzyed enzyme used for removal RNA for RNA free purification reaction of DNA and degradation of RNA into small components.
• It is used for plasmid DNA purification and genomic DNA purification as well as RNA remove from recombinant protein preparation.
• For the removal of existing RNA into the cell is achieved by degradation of RNA which is released from cell by RNase. For this it will added in PD1 buffer.

Que : Alcohol was added to the column wash buffer why? What does this do the chromosomal DNA?

• during experiment of DNA extraction, due to binding a buffer solution (ethanol) along with sample. After centrifugation the column again with forcing the wash buffer through the membrane of cell .
• It's remove the remaining impurities from the membrane of cell and leaving only DNA molecule which bind with silica gel.
• Ethanol play important roles in DNA extraction.
• By the alcohol is added it will generate attraction between phospahte group and positive ions present in solution which becomes strong to form stable ionic bond and DNA precipitation. This is occur because of DNA having phospahte back bone with highly charged polar group.

Que : name two methods of detecting DNA isolation in this week's lab?

• Detecting DNA isolation is done by this two methods

1. Solid phase extraction
2. Organic extraction

• By using this two methods isolated the DNA on the basis of quality and quantity of DNA produced. Which includes basis four steps : lysis of the sample , precipitation, washing, resuspension.

Que : Explain how detergent are used in PD2 buffer?

• For the DNA precipitation detergent are used in PD2 buffer
• During this process detergent move to the cell membrane and break down of the molecules present in sample. it mean as cell will lysis or open that will release DNA into sample solution.

Que : what structure collect the DNA by binding to the matrix?

• During the solid phase extraction , the DNA molecule in the sample is bind with solid phase of silica gel membrane to the column matrix.
• During the lysis of sample , it will broken the cell and release DNA from the cell membrane and nucleus.