Post 1: DNA Extraction

Hi All,

I realize that June is almost over and I haven’t posted any updates about my research. June has been a very busy month for me.  But, the bulk of my work has centered around the extraction of bacterial DNA from the 0.22 micron filters that are used to collect the bacteria from Lake Matoaka on a monthly basis. So far this month, I’ve extracted nine months (and counting) worth of DNA from filters that have been stored at -80 C for almost a year now. 

The extraction protocol is a time consuming one that usually takes me two days to complete per sample. It combines both physical and chemical techniques. The initial steps of the extraction process deal with breaking open the bacterial cells and releasing the cellular contents (lipids, proteins and nucleic acids). Three specific enzymes are utilized to this end. Lysozyme is an enzyme found in egg whites (and human tears) that degrades peptidoglycans, which are important molecules that compose the bacterial cell membrane. Sodium dodecyl sulfate (SDS) is a detergent that degrades lipids and Proteinase K is an enzyme that digests proteins. These three chemicals/enzymes in combination with vigorous shaking and a freeze-thaw process that stresses the cellular membrane effectively lyse open the bacterial cells captured on the sample filters.

Once the contents of the bacterial cells have been released into the extraction buffer, a series of chemicals in combination with centrifugation is used to purify the nucleic acids from the rest of the cellular contents. Phenol removes proteins and chloroform removes lipids and excess phenol from the mix of bacterial cellular contents. Finally, isopropanol is used to precipitate the nucleic acids out of the extraction buffer. Once re-suspended in stabilizing TE Buffer, the bacterial nucleic acids are ready for further use.

My next few posts will follow the progress of the extracted nucleic acids through PCR, enzyme restriction, the creation of clone libraries and sequencing. Stay tuned!

Dana