Question

. You have extracted extrachromosomal DNA and genomic DNA from E. coli in the lab. What is the differentiating steps during the DNA isolation so that your target DNA is not contaminated with the other unwanted DNA (i.e. you are targeting extrachromosomal DNA, so you don’t want the genomic DNA, and vice-versa)? Why and how that step can differentiate between these groups of DNA

Answer 1

At the time of separating extrachromosomal DNA from that of genomic DNA it should be kept in mind that chromosomal DNA is thicker than genomic DNA. The separation between the two must be based on size, and by using the correct lysis method. Chromosomal DNA can be separated using alkaline lysis method. The lysis buffer consists of sodium hydroxide and SDS which denatures the plasmid and genomic DNA. This helps in separating the DNA to single strands. Then the sample must be neutralized by the help of potassium acetate to renature the plasmid. This helps in separating plasmid DNMA to that of genomic DNA. As plasmids are small they can reanneal to form double stranded DNA. On the other hand, genomic DNA are too large and thus, they cannot reanneal and remain separated. Thus, after centrifugation genomic DNA form a pellet whereas extrachromosomal DNA remains soluble. This step will help in separating the two types of DNA preventing any contamination.