Steps of Dna Replication
Background on Genomic DNA Isolation and Purification
Generally, all methods involve the disruption and lysis of cells. This is followed sometimes by the removal of RNA (by RNAses, salt or other methods). Choosing which method to use will depend on many selection factors including:
DNA is isolated from proteins by several methods including digestion of proteins by the enzyme proteinase K. Proteins are removed subsequently by salting-out, organic extraction, or binding of the DNA to a solid-phase support (such as an anion-exchange column or silica technology).
DNA is finally recovered by ethanol precipitation or isopropanol precipitation.
In general, the separation of DNA from cells and cellular components can be divided into four stages: 1. Cell disruption 2. Lysis of Cell 3. Removal of Proteins and Contaminants 4. Recovery of DNA
In some genomic DNA isolation protocols, stages 1 and 2 are combined.
ISOLATION AND QUANTIFICATION OF GENOMIC DNA
Learning objectives for this lab:
¾ Understand the basic process behind a DNA isolation (extraction) procedure
¾ To get a “feel” for what DNA looks like
¾ To learn how to determine the concentration of DNA using spectrophotometry
¾ To learn how to make a standard curve
First: Read through the entire lab. Focus on the questions you will need to answer.
Second: With your group, sketch out a flow chart of each step. Each person should have this in their notebook. Use diagrams/short sentences.
DNA Quantification
You will also learn how to determine the concentration of the DNA you isolate using a spectrophotometer. You will measure the absorbance of the DNA solution you isolate and compare it to a DNA solution of known concentration.
DNA Isolation Protocol
1. Weigh out 60 g of onion and mince it into smaller pieces using a razor blade (carefully!). You may use the scale at the back bench to check the weight of your pieces.
2. Put your minced onion and 50 ml of the cold
Generally, all methods involve the disruption and lysis of cells. This is followed sometimes by the removal of RNA (by RNAses, salt or other methods). Choosing which method to use will depend on many selection factors including:
DNA is isolated from proteins by several methods including digestion of proteins by the enzyme proteinase K. Proteins are removed subsequently by salting-out, organic extraction, or binding of the DNA to a solid-phase support (such as an anion-exchange column or silica technology).
DNA is finally recovered by ethanol precipitation or isopropanol precipitation.
In general, the separation of DNA from cells and cellular components can be divided into four stages: 1. Cell disruption 2. Lysis of Cell 3. Removal of Proteins and Contaminants 4. Recovery of DNA
In some genomic DNA isolation protocols, stages 1 and 2 are combined.
ISOLATION AND QUANTIFICATION OF GENOMIC DNA
Learning objectives for this lab:
¾ Understand the basic process behind a DNA isolation (extraction) procedure
¾ To get a “feel” for what DNA looks like
¾ To learn how to determine the concentration of DNA using spectrophotometry
¾ To learn how to make a standard curve
First: Read through the entire lab. Focus on the questions you will need to answer.
Second: With your group, sketch out a flow chart of each step. Each person should have this in their notebook. Use diagrams/short sentences.
DNA Quantification
You will also learn how to determine the concentration of the DNA you isolate using a spectrophotometer. You will measure the absorbance of the DNA solution you isolate and compare it to a DNA solution of known concentration.
DNA Isolation Protocol
1. Weigh out 60 g of onion and mince it into smaller pieces using a razor blade (carefully!). You may use the scale at the back bench to check the weight of your pieces.
2. Put your minced onion and 50 ml of the cold