Isolating Genomic DNA

Isolating genomic DNA Introduction and history Prior to 1944 it was thought the genetic material must surely be protein. This reasoning was that DNA was too dull, with its four repetitious bases, to be of much use in heredity Proteins, on the other hand were enormously complex and varied, and could probably handle the complicated demands of heredity To study the biological, chemical, and physical properties of DNA it was necessary to obtain the material in a native, highly polymerized state Basics of isolating DNA Applications In BMB 442, we are going to isolate chromosomal DNA from Escherichia coli (E. coli) in order to clone a gene that expresses the protein alkaline phosphates In order for PCR to work efficiently, it is desirable to purify the longest DNA fragments possible and be free of contaminants which might interfere in the reaction What is genomic DNA? Genomic DNA can be defined as the organisms complete set of DNA The E. coli cells that we are using, HB101, do not have extrachromosomal DNA (i.e. Plasmids), the reform our experiment is only isolating chromosomal DNA There are five basic steps to isolating genomic DNA: (1) sample collection (2) cell lysis (3) purification of DNA from cellular debris (4) collection of DNA (5) quantification and assessment of DNA qualify Sample collection The first step of any DNA extraction is to collect the cells from which you are isolating DNA. Our protocol is fairly straightforward since we will grow a culture of HB101 E. coli cells and collect them by centrifugation Cell lysis Breaking open the cell or cell lysis is the second step. Inefficient lysis leads to less DNA collected and harsh lysis can sometimes damage the DNA Purification of DNA from cellular debris The next step is to remove cellular debris such as proteins, RNA, lipids, etc. from the DNA Chloroform isoamyl-alcohol RNA and DNA Precipitated and denatured proteins unguentum Lipids Collection of DNA At this point in our procedure, the DNA would be in 30 mL of liquid and we are going to remove the DNA from the 30 mL by precipitation and dissolve it in 4 ml of water to concentrate it Quantification and assessment of DNA quality The final step of our experiment is to determine how much DNA we isolated and whether it is pure or contaminated with cellular debris A260/A280. Quality Ratio > 2.0. RNA contamination 1.8 < ratio < 2.0. Pure DNA Ratio < 1.8. Protein contamination