Week 11- Turning up the heat

This week I began constructing the rough draft of my research paper. While incomplete, the formatting has been done and the paper is ready for insertion of data as the project progresses. Given the nature and pace of the project thus far, I anticipate final completion of the project to occur in the fall semester. That said, I have finished the initial phase of the project, which dealt with testing various DNA extraction protocols. I tested four protocols that utilized boiling, digestion, and cold ethanol. The simple boiling methods worked best not only at extraction, but were also preferable for ease of use. As one of the goals of my project is to develop a micro lab that will introduce the student learner to elementary DNA identification, it is important that I identify a protocol that is both simplified and time-efficient, so that the method will fit in the typical allotted class time.

This method was both easiest to use and successful at extracting Escherichia coli DNA:

 

Protocol 1: Autoclave 200 mL of de-ionized (DI) water on media cycle @ 121°C/15 PSI. Centrifuge 400µl of E. coli culture @ 10,000x g for 10 minutes to pellet the sample. Re-suspend pellet in 100µl of autoclaved DI water. Incubate for 15 minutes @ 100°C in hot plate bath. Centrifuge sample @ 10,000x g for 10 minutes. Remove supernatant and place into new Eppendorf tube. Store prepared sample @-20°C until ready to use.

This protocol had the added benefit of teaching the student sterile technique, due to the utilization not only of the autoclave, as in P1, but also the use of molecular-biology grade water:

Protocol 4: Autoclave beaker on media cycle @121°C/15PSI to sterilize. Add 1ml of E. coli to 1.5 ml Eppendorf. Centrifuge @13,200x g for 15 minutes. Eliminate supernatant, and re-suspend pellets in m-b grade water. Centrifuge @13,200x g for 10 minutes. Eliminate supernatant, and re-suspend pellets in 40µl of m-b- grade water. Boil @100°C for 10 minutes. Cool on ice. Centrifuge @13,200x g for 10 seconds.

Today, I begin DNA amplification via the PCR process, so I will begin testing the universality of the primers I chose for this project. More on that next week. Cheers!
Here's a PCR machine in the old days, and a link to its origin and some useful information:
http://www.plantcellbiology.com/2012/03/you-dont-know-how-lucky-you-are-pcr-in-the-old-days/